Nucleic acids encoding human CIDE-B protein and polymorphic markers thereof

ABSTRACT

The present invention relates to a purified or isolated polynucleotide encoding human CIDE B protein, the regulatory nucleic acids contained therein, polymorphic markers thereof, and the resulting encoded protein, as well as to methods and kits for detecting this polynucleotide and this protein. The present invention also pertains to a polynucleotide carrying the natural regulatory regions of the CIDE B gene which is useful, for example, to express a heterologous nucleic acid in host cells or host organisms as well as functionally active regulatory polynucleotides derived from said regulatory regions.

FIELD OF THE INVENTION

[0001] The present invention relates to a purified or isolatedpolynucleotide encoding human CIDE B protein, the regulatory nucleicacids contained therein, polymorphic markers thereof, and the resultingencoded protein, as well as to methods and kits for detecting thispolynucleotide and this protein. The present invention also pertains toa polynucleotide carrying the natural regulatory regions of the CIDE Bgene which is useful, for example, to express a heterologous nucleicacid in host cells or host organisms as well as functionally activeregulatory polynucleotides derived from said regulatory regions.

BACKGROUND OF THE INVENTION

[0002] Apoptosis is of fundamental importance to biological processesincluding embryogenesis, maintenance of tissue homeostasis, normalcellular development of multicellular organisms, elimination ofvirus-infected cells, and the development of immune system. It is a typeof death that is fundamentally distinct from degenerative death ornecrosis in that it is an active process of gene-directed cellularself-destruction which, in some instances, serves a biologicallymeaningful homeostatic function. Necrosis, in contrast, is cell deathoccurring as a result of severe injurious changes in the environment ofinfected cells.

[0003] Morphologically, apoptosis is characterized by the rapidcondensation of the cell with preservation of membranes.Synchronistically with the compaction of chromatin, several biochemicalchanges occur in the cell. Nuclear DNA is cleaved at the linker regionsbetween nucleosomes to produce fragments that are easily demonstrated byagarose gel electrophoresis wherein a characteristic ladder develops.

[0004] The primary image of apoptosis is that of the dying thymocyte:fusion of chromatin into one mass, which binds to the nuclear membrane,while the cytoplasm remains apparently intact before beginning tocondense. The nuclear change is one of the earliest visible processes;the conversion to the condensed state occurs rapidly and is accompaniedby endonucleolytic degradation of DNA between nucleosomes. Once thechromatin has condensed, electrophoresis of the DNA demonstrates aladder of fragments differing in size by 180 bp, generated by anenzymatic activity resembling that of DNase I.

[0005] This type of cell death is seen in many varieties of cells,especially those that, like lymphocytes or thymocytes, have relativelylittle cytoplasm and are highly mitotic or derive from highly mitoticlines. In this situation, in which mitotic cells are likely to facechallenges by mutagens (viruses, toxins), an appropriate biologicalimperative would be to destroy the DNA rapidly and effectively. Thus,this type of cell death is particularly dramatic among hematopoieticcells and their derivatives.

[0006] Several regulatory components of the apoptotic pathway have beenidentified in various living organisms including man and the nematodeCaenorhabditis elegans.

[0007] Two murine transcription products involved in cell apoptosis havebeen reported by Inohara et al. (1998), that have been namedrespectively CIDE-A and CIDE-B. Murine CIDE-A and CIDE-B have stronghomology with the murine anti-apoptosis DFF45 protein as well as withthe drosophila protein DREP-1. The homology of CIDE-A, CIDE-B and FSP27with DFF45 was restricted to an N-terminal region designated by Inoharaet al. as CIDE-N domain which showed 39, 29 and 38% amino acid identityrespectively with DFF45.

[0008] Because there is a strong need in the art to make available tothe public novel means useful to prevent or inhibit apoptosis disorders,either in the case of disorders caused by abnormal cell proliferationwherein apoptosis induction is desirable or in the case of disorderscaused by abnormal cell death wherein an inhibition or an arrest ofapoptosis is desirable, the inventors have attempted to isolate andcharacterize a novel gene encoding a protein involved in apoptosispathway, namely the human CIDE-B gene.

SUMMARY OF THE INVENTION

[0009] The present invention pertains to a nucleic acid moleculecomprising the genomic sequence of the human CIDE B gene. The CIDE Bgenonic sequence comprises regulatory sequences located both upstream(5′-end) and downstream (3′-end) of the transcribed portion of saidgene, these regulatory sequences being also part of the invention.

[0010] The invention also deals with the complete cDNA sequence encodingthe CIDE B protein, as well as with the corresponding translationproduct.

[0011] Oligonucleotide probes or primers hybridizing specifically with aCIDE B genomic or cDNA sequence are also part of the present invention.

[0012] A further object of the invention consists of recombinant vectorscomprising any of the nucleic acid sequences above described, and inparticular of recombinant vectors comprising a CIDE B regulatorysequence or a sequence encoding a CIDE B protein, as well as of cellhosts comprising said nucleic acid sequences or recombinant vectors.

[0013] Finally, the invention is directed to methods for the screeningof substances or molecules which modulate the expression of CIDE B.

[0014] The invention is also directed to biallelic markers that arelocated within the CIDE B genomic sequence, these biallelic markersrepresenting useful tools in order to identify a statisticallysignificant association between specific alleles of CIDE B gene and oneor several disorders related to apoptosis such as cancer and AIDS.

BRIEF DESCRIPTION OF THE DRAWING

[0015]FIG. 1 is a diagram of the CIDE B1 gene with an indication of therelative position of the biallelic markers of the present invention. Theupper line refers to the genomic sequence of CIDE B. The middle linerefers to the cDNA. The lower line refers to the CIDE B protein.

BRIEF DESCRIPTION OF THE SEQUENCES PROVIDED IN THE SEQUENCE LISTING

[0016] SEQ ID No 1 contains a genomic sequence of CIDE B comprising the5′ regulatory region (upstream untranscribed region), the exons andintrons, and the 3′ regulatory region (downstream untranscribed region).

[0017] SEQ ID No 2 contains a cDNA sequence of CIDE B.

[0018] SEQ ID No 3 contains the amino acid sequence encoded by the cDNAof SEQ ID No 2.

[0019] SEQ ID No 4 contains a primer containing the additional PU 5′sequence described further in Example 2

[0020] SEQ ID No 5 contains a primer containing the additional RP 5′sequence described further in Example 2.

[0021] In accordance with the regulations relating to Sequence Listings,the following codes have been used in the Sequence Listing to indicatethe locations of biallelic markers within the sequences and to identifyeach of the alleles present at the polymorphic base. The code “r” in thesequences indicates that one allele of the polymorphic base is aguanine, while the other allele is an adenine. The code “y” in thesequences indicates that one allele of the polymorphic base is athymine, while the other allele is a cytosine. The code “m” in thesequences indicates that one allele of the polymorphic base is anadenine, while the other allele is an cytosine. The code “k” in thesequences indicates that one allele of the polymorphic base is aguanine, while the other allele is a thymine. The code “s” in thesequences indicates that one allele of the polymorphic base is aguanine, while the other allele is a cytosine. The code “w” in thesequences indicates that one allele of the polymorphic base is anadenine, while the other allele is an thymine. The nucleotide code ofthe original allele for each biallelic marker is the following:Biallelic marker Original allele 12-73-49 C 12-74-38 T

DETAILED DESCRIPTION OF THE INVENTION

[0022] The aim of the present invention is to provide the human CIDE Bgene, the human CIDE B mRNA molecules and the polynucleotides derivedfrom them. The polynucleotides of the invention are useful to designsuitable means for detecting the presence of this gene or cDNA in a testsample and to design suitable means to express a desired polynucleotideof interest. The invention also relates to the human CIDE B polypeptide.

Definitions

[0023] Before describing the invention in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used to describe the invention herein.

[0024] The terms “CIDE B gene”, when used herein, encompasses genomic,mRNA and cDNA sequences encoding the CIDE B protein.

[0025] The term “heterologous protein”, when used herein, is intended todesignate any protein or polypeptide other than the CIDE B protein. Moreparticularly, the heterologous protein is a compound which can be usedas a marker in further experiments with a CIDE B regulatory region.

[0026] The term “isolated” requires that the material be removed fromits original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally-occurring polynucleotideor polypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat the vector or composition is not part of its natural environment.

[0027] The term “purified” does not require absolute purity; rather, itis intended as a relative definition. Purification of starting materialor natural material to at least one order of magnitude, preferably twoor three orders, and more preferably four or five orders of magnitude isexpressly contemplated. As an example, purification from 0.1%concentration to 10% concentration is two orders of magnitude. The term“purified” is used herein to describe a polynucleotide or polynucleotidevector of the invention which has been separated from other compoundsincluding, but not limited to other nucleic acids, carbohydrates, lipidsand proteins (such as the enzymes used in the synthesis of thepolynucleotide), or the separation of covalently closed polynucleotidesfrom linear polynucleotides. A polynucleotide is substantially pure whenat least about 50%, preferably 60 to 75% of a sample exhibits a singlepolynucleotide sequence and conformation (linear versus covalentlyclose). A substantially pure polynucleotide typically comprises about50%, preferably 60 to 90% weight/weight of a nucleic acid sample, moreusually about 95%, and preferably is over about 99% pure. Polynucleotidepurity or homogeneity is indicated by a number of means well known inthe art, such as agarose or polyacrylamide gel electrophoresis of asample, followed by visualizing a single polynucleotide band uponstaining the gel. For certain purposes higher resolution can be providedby using HPLC or other means well known in the art.

[0028] The term “polypeptide” refers to a polymer of amino acids withoutregard to the length of the polymer; thus, peptides, oligopeptides, andproteins are included within the definition of polypeptide. This termalso does not specify or exclude post-expression modifications ofpolypeptides, for example, polypeptides which include the covalentattachment of glycosyl groups, acetyl groups, phosphate groups, lipidgroups and the like are expressly encompassed by the term polypeptide.Also included within the definition are polypeptides which contain oneor more analogs of an amino acid (including, for example, non-naturallyoccurring amino acids, amino acids which only occur naturally in anunrelated biological system, modified amino acids from mammalian systemsetc.), polypeptides with substituted linkages, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring.

[0029] The term “recombinant polypeptide” is used herein to refer topolypeptides that have been artificially designed and which comprise atleast two polypeptide sequences that are not found as contiguouspolypeptide sequences in their initial natural environment, or to referto polypeptides which have been expressed from a recombinantpolynucleotide.

[0030] The term “purified” is used herein to describe a polypeptide ofthe invention which has been separated from other compounds including,but not limited to nucleic acids, lipids, carbohydrates and otherproteins. A polypeptide is substantially pure when at least about 50%,preferably 60 to 75% of a sample exhibits a single polypeptide sequence.A substantially pure polypeptide typically comprises about 50%,preferably 60 to 90% weight/weight of a protein sample, more usuallyabout 95%, and preferably is over about 99% pure. Polypeptide purity orhomogeneity is indicated by a number of means well known in the art,such as polyacrylamide gel electrophoresis of a sample, followed byvisualizing a single polypeptide band upon staining the gel. For certainpurposes higher resolution can be provided by using HPLC or other meanswell known in the art.

[0031] As used herein, the term “non-human animal” refers to anynon-human vertebrate, birds and more usually mammals, preferablyprimates, farm animals such as swine, goats, sheep, donkeys, and horses,rabbits or rodents, more preferably rats or mice. As used herein, theterm “animal” is used to refer to any vertebrate, preferable a mammal.Both the terms “animal” and “mammal” expressly embrace human subjectsunless preceded with the term “non-human”.

[0032] As used herein, the term “antibody” refers to a polypeptide orgroup of polypeptides which are comprised of at least one bindingdomain, where an antibody binding domain is formed from the folding ofvariable domains of an antibody molecule to form three-dimensionalbinding spaces with an internal surface shape and charge distributioncomplementary to the features of an antigenic determinant of an antigen,which allows an immunological reaction with the antigen. Antibodiesinclude recombinant proteins comprising the binding domains, as wells asfragments, including Fab, Fab′, F(ab)₂, and F(ab′)₂ fragments.

[0033] As used herein, an “antigenic determinant” is the portion of anantigen molecule, in this case a CIDE B polypeptide, that determines thespecificity of the antigen-antibody reaction. An “epitope” refers to anantigenic determinant of a polypeptide. An epitope can comprise as fewas 3 amino acids in a spatial conformation which is unique to theepitope. Generally an epitope comprises at least 6 such amino acids, andmore usually at least 8-10 such amino acids. Methods for determining theamino acids which make up an epitope include x-ray crystallography,2-dimensional nuclear magnetic resonance, and epitope mapping e.g. thePepscan method described by Geysen et al. 1984; PCT Publication No. WO84/03564; and PCT Publication No. WO 84/03506.

[0034] Throughout the present specification, the expression “nucleotidesequence” may be employed to designate indifferently a polynucleotide ora nucleic acid. More precisely, the expression “nucleotide sequence”encompasses the nucleic material itself and is thus not restricted tothe sequence information (i.e. the succession of letters chosen amongthe four base letters) that biochemically characterizes a specific DNAor RNA molecule.

[0035] As used interchangeably herein, the terms “nucleic acids”,“oligonucleotides”, and “polynucleotides” include RNA, DNA, or RNA/DNAhybrid sequences of more than one nucleotide in either single chain orduplex form. The term “nucleotide” as used herein as an adjective todescribe molecules comprising RNA, DNA, or RNA/DNA hybrid sequences ofany length in single-stranded or duplex form. The term “nucleotide” isalso used herein as a noun to refer to individual nucleotides orvarieties of nucleotides, meaning a molecule, or individual unit in alarger nucleic acid molecule, comprising a purine or pyrimidine, aribose or deoxyribose sugar moiety, and a phosphate group, orphosphodiester linkage in the case of nucleotides within anoligonucleotide or polynucleotide. Although the term “nucleotide” isalso used herein to encompass “modified nucleotides” which comprise atleast one modifications (a) an alternative linking group, (b) ananalogous form of purine, (c) an analogous form of pyrimidine, or (d) ananalogous sugar, for examples of analogous linking groups, purine,pyrimidines, and sugars see for example PCT publication No. WO 95/04064.The polynucleotide sequences of the invention may be prepared by anyknown method, including synthetic, recombinant, ex vivo generation, or acombination thereof, as well as utilizing any purification methods knownin the art.

[0036] A “promoter” refers to a DNA sequence recognized by the syntheticmachinery of the cell required to initiate the specific transcription ofa gene.

[0037] A sequence which is “operably linked” to a regulatory sequencesuch as a promoter means that said regulatory element is in the correctlocation and orientation in relation to the nucleic acid to control RNApolymerase initiation and expression of the nucleic acid of interest.

[0038] As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the coding sequence. More precisely, twoDNA molecules (such as a polynucleotide containing a promoter region anda polynucleotide encoding a desired polypeptide or polynucleotide) aresaid to be “operably linked” if the nature of the linkage between thetwo polynucleotides does not (1) result in the introduction of aframe-shift mutation or (2) interfere with the ability of thepolynucleotide containing the promoter to direct the transcription ofthe coding polynucleotide.

[0039] The term “primer” denotes a specific oligonucleotide sequencewhich is complementary to a target nucleotide sequence and used tohybridize to the target nucleotide sequence. A primer serves as aninitiation point for nucleotide polymerization catalyzed by either DNApolymerase, RNA polymerase or reverse transcriptase.

[0040] The term “probe” denotes a defined nucleic acid segment (ornucleotide analog segment, e.g., polynucleotide as defined hereinbelow)which can be used to identify a specific polynucleotide sequence presentin samples, said nucleic acid segment comprising a nucleotide sequencecomplementary of the specific polynucleotide sequence to be identified.

[0041] The terms “trait” and “phenotype” are used interchangeably hereinand refer to any visible, detectable or otherwise measurable property ofan organism such as symptoms of, or susceptibility to a disease forexample. Typically the terms “trait” or “phenotype” are used herein torefer to symptoms of, or susceptibility to a disease, a beneficialresponse to or side effects related to a treatment. Preferably, saidtrait can be, without to be limited to, cancers, developmental diseases,and neurological diseases.

[0042] The term “allele” is used herein to refer to variants of anucleotide sequence. A biallelic polymorphism has two forms. Diploidorganisms may be homozygous or heterozygous for an allelic form.

[0043] The term “genotype” as used herein refers the identity of thealleles present in an individual or a sample. In the context of thepresent invention, a genotype preferably refers to the description ofthe biallelic marker alleles present in an individual or a sample. Theterm “genotyping” a sample or an individual for a biallelic markerinvolves determining the specific allele or the specific nucleotidecarried by an individual at a biallelic marker.

[0044] The term “mutation” as used herein refers to a difference in DNAsequence between or among different genomes or individuals which has afrequency below 1%.

[0045] The term “polymorphism” as used herein refers to the occurrenceof two or more alternative genomic sequences or alleles between or amongdifferent genomes or individuals. “Polymorphic” refers to the conditionin which two or more variants of a specific genomic sequence can befound in a population. A “polymorphic site” is the locus at which thevariation occurs. A single nucleotide polymorphism is the replacement ofone nucleotide by another nucleotide at the polymorphic site. Deletionof a single nucleotide or insertion of a single nucleotide also givesrise to single nucleotide polymorphisms. In the context of the presentinvention, “single nucleotide polymorphism” preferably refers to asingle nucleotide substitution. Typically, between differentindividuals, the polymorphic site may be occupied by two differentnucleotides.

[0046] The term “biallelic polymorphism” and “biallelic marker” are usedinterchangeably herein to refer to a single nucleotide polymorphismhaving two alleles at a fairly high frequency in the population. A“biallelic marker allele” refers to the nucleotide variants present at abiallelic marker site.

[0047] The location of nucleotides in a polynucleotide with respect tothe center of the polynucleotide are described herein in the followingmanner. When a polynucleotide has an odd number of nucleotides, thenucleotide at an equal distance from the 3′ and 5′ ends of thepolynucleotide is considered to be “at the center” of thepolynucleotide, and any nucleotide immediately adjacent to thenucleotide at the center, or the nucleotide at the center itself isconsidered to be “within 1 nucleotide of the center.” With an odd numberof nucleotides in a polynucleotide any of the five nucleotides positionsin the middle of the polynucleotide would be considered to be within 2nucleotides of the center, and so on. When a polynucleotide has an evennumber of nucleotides, there would be a bond and not a nucleotide at thecenter of the polynucleotide. Thus, either of the two centralnucleotides would be considered to be “within 1 nucleotide of thecenter” and any of the four nucleotides in the middle of thepolynucleotide would be considered to be “within 2 nucleotides of thecenter”, and so on.

[0048] Biallelic markers can be defined as genome-derivedpolynucleotides having between 2 and 100, preferably between 20, 30, or40 and 60, and more preferably about 47 nucleotides in length, whichexhibit biallelic polymorphism at one single base position. Eachbiallelic marker therefore corresponds to two forms of a polynucleotidesequence included in a gene which, when compared with one another,present a nucleotide modification at one position.

[0049] The term “upstream” is used herein to refer to a location whichis toward the 5′ end of the polynucleotide from a specific referencepoint.

[0050] The terms “base paired” and “Watson & Crick base paired” are usedinterchangeably herein to refer to nucleotides which can be hydrogenbonded to one another be virtue of their sequence identities in a mannerlike that found in double-helical DNA with thymine or uracil residueslinked to adenine residues by two hydrogen bonds and cytosine andguanine residues linked by three hydrogen bonds (See Stryer, 1995).

[0051] The terms “complementary” or “complement thereof” are used hereinto refer to the sequences of polynucleotides which is capable of formingWatson & Crick base pairing with another specified polynucleotidethroughout the entirety of the complementary region. For the purpose ofthe present invention, a first polynucleotide is deemed to becomplementary to a second polynucleotide when each base in the firstpolynucleotide is paired with its complementary base. Complementarybases are, generally, A and T (or A and U), or C and G. “Complement” isused herein as a synonym from “complementary polynucleotide”,“complementary nucleic acid” and “complementary nucleotide sequence”.These terms are applied to pairs of polynucleotides based solely upontheir sequences and not any particular set of conditions under which thetwo polynucleotides would actually bind.

[0052] Variants and Fragments

[0053] 1-Polynucleotides

[0054] The invention also relates to variants and fragments of thepolynucleotides described herein.

[0055] Variants of polynucleotides, as the term is used herein, arepolynucleotides that differ from a reference polynucleotide. A variantof a polynucleotide may be a naturally occurring variant such as anaturally occurring allelic variant, or it may be a variant that is notknown to occur naturally. Such non-naturally occurring variants of thepolynucleotide may be made by mutagenesis techniques, including thoseapplied to polynucleotides, cells or organisms. Generally, differencesare limited so that the nucleotide sequences of the reference and thevariant are closely similar overall and, in many regions, identical.

[0056] Nucleotide changes present in a variant polynucleotide may besilent, which means that they do not alter the amino acids encoded bythe polynucleotide.

[0057] However, nucleotide changes may also result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence. The substitutions,deletions or additions may involve one or more nucleotides. The variantsmay be altered in coding or non-coding regions or both. Alterations inthe coding regions may produce conservative or non-conservative aminoacid substitutions, deletions or additions.

[0058] In the context of the present invention, particularly preferredembodiments are those in which the polynucleotides encode polypeptideswhich retain substantially the same biological function or activity asthe mature CIDE B protein.

[0059] Variants of polynucleotides according to the invention include,without being limited to, nucleotide sequences at least 95% identical toa nucleic acid selected from the group consisting of SEQ ID Nos 1 and 2,and any polynucleotide fragment of at least 8, 20, 50, 75, or 100consecutive nucleotides from a nucleic acid selected from the groupconsisting of SEQ ID Nos 1 and 2, and preferably at least 99% identical,more particularly at least 99.5% identical, and most preferably at least99.8% identical to a nucleic acid selected from the group consisting ofSEQ ID Nos 1 and 2, and any polynucleotide fragment of at least 8, 20,50, 75, or 100 consecutive nucleotides from a nucleic acid selected fromthe group consisting of SEQ ID Nos 1 and 2.

[0060] A polynucleotide fragment is a polynucleotide having a sequencethat is entirely the same as part but not all of a given nucleotidesequence, preferably the nucleotide sequence of a CIDE B gene, andvariants thereof. The fragment can be a portion of an exon or of anintron of a CIDE B gene. It can also be a portion of the regulatorysequences of the CIDE B gene, preferably of the promoter. Preferably,such fragments comprise at least one of the biallelic markers 12-73-49and 12-74-38 or a biallelic marker in linkage disequilibrium therewith.

[0061] Such fragments may be “free-standing”, i.e. not part of or fusedto other polynucleotides, or they may be comprised within a singlelarger polynucleotide of which they form a part or region. However,several fragments may be comprised within a single largerpolynucleotide.

[0062] As representative examples of polynucleotide fragments of theinvention, there may be mentioned those which have from about 4, 6, 8,15, 20, 25, 40, 10 to 30, 30 to 55, 50 to 100, 75 to 100 or 100 to 200nucleotides in length. Preferred are those fragments having about 47nucleotides in length, such as those comprising one of the biallelicmarkers 12-73-49 and 12-74-38. Preferred polynucleotide fragmentsaccording to the invention comprise a contiguous span of at least 35,40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of oneparticular nucleic acid. 2-Polypeptides

[0063] The invention also relates to variants, fragments, analogs andderivatives of the polypeptides described herein, including mutated CIDEB proteins.

[0064] The variant may be 1) one in which one or more of the amino acidresidues are substituted with a conserved or non-conserved amino acidresidue and such substituted amino acid residue may or may not be oneencoded by the genetic code, or 2) one in which one or more of the aminoacid residues includes a substituent group, or 3) one in which themutated CIDE B is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol), or 4) one in which the additional amino acids are fused to themutated CIDE B, such as a leader or secretory sequence or a sequencewhich is employed for purification of the mutated CIDE B or a preproteinsequence. Such variants are deemed to be within the scope of thoseskilled in the art.

[0065] More particularly, a variant CIDE B polypeptide comprises aminoacid changes ranging from 1, 2, 3, 4, 5, 10 to 20 substitutions,additions or deletions of one amino acid, preferably from 1 to 10, morepreferably from 1 to 5 and most preferably from 1 to 3 substitutions,additions or deletions of one amino acid. The preferred amino acidchanges are those which have little or no influence on the biologicalactivity or the capacity of the variant CIDE B polypeptide to berecognized by antibodies raised against a native CIDE B protein.

[0066] By homologous peptide according to the present invention is meanta polypeptide containing one or several aminoacid additions, deletionsand/or substitutions in the amino acid sequence of a CIDE B polypeptide.In the case of an aminoacid substitution, one or several—consecutive ornon-consecutive aminoacids are replaced by <<equivalent>> amino acids.

[0067] In the case of an amino acid substitution in the amino acidsequence of a polypeptide according to the invention, one or severalamino acids can be replaced by “equivalent” amino acids. The expression“equivalent” amino acid is used herein to designate any amino acid thatmay be substituted for one of the amino acids having similar properties,such that one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the polypeptide to besubstantially unchanged. Generally, the following groups of amino acidsrepresent equivalent changes: (1) Ala, Pro, Gly, Glu, Asp, Gln, Asn,Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val, Ile, Leu, Met, Ala, Phe; (4)Lys, Arg, His; (5) Phe, Tyr, Trp, His.

[0068] A specific embodiment of a modified CIDE B peptide molecule ofinterest according to the present invention, includes, but is notlimited to, a peptide molecule which is resistatnt to proteolysis, is apeptide in which the —CONH— peptide bond is modified and replaced by a(CH2NH) reduced bond, a (NHCO) retro inverso bond, a (CH2-O)methylene-oxy bond, a (CH2-S) thiomethylene bond, a (CH2CH2) carba bond,a (CO—CH2) cetomethylene bond, a (CHOH—CH2) hydroxyethylene bond), a(N—N) bound, a E-alcene bond or also a —CH═CH— bond. The invention alsoencompasses a human CIDE B polypeptide or a fragment or a variantthereof in which at least one peptide bond has been modified asdescribed above.

[0069] The polypeptide according to the invention could havepost-translational modifications. For example, it can present thefollowing modifications: acylation, disulfide bond formation,prenylation, carboxymethylation and phosphorylation.

[0070] A polypeptide fragment is a polypeptide having a sequence thatentirely is the same as part but not all of a given polypeptidesequence, preferably a polypeptide encoded by a CIDE B gene and variantsthereof.

[0071] Such fragments may be “free-standing”, i.e. not part of or fusedto other polypeptides, or they may be comprised within a single largerpolypeptide of which they form a part or region. However, severalfragments may be comprised within a single larger polypeptide.

[0072] As representative examples of polypeptide fragments of theinvention, there may be mentioned those which have from about 5, 6, 7,8, 9 or 10 to 15, 10 to 20, 15 to 40, or 30 to 55 amino acids long.Preferred polypeptide fragments according to the invention comprise acontiguous span of at least 8 amino acids, preferably at least 10 aminoacids, more preferably at least 12, 15, 20, 25, 30, 40, 50, 100, 150 or200 amino acids of one amino acid sequence. Preferred are thosefragments containing at least one amino acid mutation in the CIDE Bprotein.

[0073] Identity between Nucleic Acids or Polypeptides

[0074] The terms “percentage of sequence identity” and “percentagehomology” are used interchangeably herein to refer to comparisons amongpolynucleotides and polypeptides, and are determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity. Homology is evaluated using any of the variety of sequencecomparison algorithms and programs known in the art. Such algorithms andprograms include, but are by no means limited to, TBLASTN, BLASTP,FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988; Altschul et al.,1990; Thompson et al., 1994; Higgins et al., 1996; Altschul et al.,1990; Altschul et al., 1993). In a particularly preferred embodiment,protein and nucleic acid sequence homologies are evaluated using theBasic Local Alignment Search Tool (“BLAST”) which is well known in theart (see, e.g., Karlin and Altschul, 1990; Altschul et al., 1990, 1993,1997). In particular, five specific BLAST programs are used to performthe following task:

[0075] (1) BLASTP and BLAST3 compare an amino acid query sequenceagainst a protein sequence database;

[0076] (2) BLASTN compares a nucleotide query sequence against anucleotide sequence database;

[0077] (3) BLASTX compares the six-frame conceptual translation productsof a query nucleotide sequence (both strands) against a protein sequencedatabase;

[0078] (4) TBLASTN compares a query protein sequence against anucleotide sequence database translated in all six reading frames (bothstrands); and

[0079] (5) TBLASTX compares the six-frame translations of a nucleotidequery sequence against the six-frame translations of a nucleotidesequence database.

[0080] The BLAST programs identify homologous sequences by identifyingsimilar segments, which are referred to herein as “high-scoring segmentpairs,” between a query amino or nucleic acid sequence and a testsequence which is preferably obtained from a protein or nucleic acidsequence database. High-scoring segment pairs are preferably identified(i.e., aligned) by means of a scoring matrix, many of which are known inthe art. Preferably, the scoring matrix used is the BLOSUM62 matrix(Gonnet et al., 1992; Henikoff and Henikoff, 1993). Less preferably, thePAM or PAM250 matrices may also be used (see, e.g., Schwartz andDayhoff, eds., 1978). The BLAST programs evaluate the statisticalsignificance of all high-scoring segment pairs identified, andpreferably selects those segments which satisfy a user-specifiedthreshold of significance, such as a user-specified percent homology.Preferably, the statistical significance of a high-scoring segment pairis evaluated using the statistical significance formula of Karlin (see,e.g., Karlin and Altschul, 1990).

[0081] The BLAST programs may be used with the default parameters orwith modified parameters provided by the user.

Stringent Hybridization Conditions

[0082] By way of example and not limitation, procedures using conditionsof high stringency are as follows: Prehybridization of filterscontaining DNA is carried out for 8 h to overnight at 65° C. in buffercomposed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02%Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters arehybridized for 48 h at 65° C., the preferred hybridization temperature,in prehybridization mixture containing 100 μg/ml denatured salmon spermDNA and 5-20×10⁶ cpm of ³²P-labeled probe. Alternatively, thehybridization step can be performed at 65° C. in the presence of SSCbuffer, 1×SSC corresponding to 0.15M NaCl and 0.05 M Na citrate.Subsequently, filter washes can be done at 37° C. for 1 h in a solutioncontaining 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by awash in 0.1×SSC at 50° C. for 45 min. Alternatively, filter washes canbe performed in a solution containing 2×SSC and 0.1% SDS, or 0.5×SSC and0.1% SDS, or 0.1×SSC and 0.1% SDS at 68° C. for 15 minute intervals.Following the wash steps, the hybridized probes are detectable byautoradiography. Other conditions of high stringency which may be usedare well known in the art and cited in Sambrook et al., 1989; andAusubel et al., 1989. These hybridization conditions are suitable for anucleic acid molecule of about 20 nucleotides in length. There is noneed to say that the hybridization conditions described above are to beadapted according to the length of the desired nucleic acid, followingtechniques well known to the one skilled in the art. The suitablehybridizations conditions may for example be adapted according to theteachings disclosed in the book Hames and Higgins (1985) or in Sambrooket al.(1989).

Genomic Sequences of CIDE B

[0083] The present invention comprises a purified or isolated nucleicacid encoding the CIDE B polypeptide, wherein said nucleic acidcomprising the sequence of SEQ ID No 1, a sequence complementarythereto, a fragment or a variant thereof.

[0084] The invention also encompasses a purified or isolated nucleicacid comprising a nucleotide sequence having at least 70, 75, 80, 85,90, or 95% nucleotide identity with the nucleotide sequence of SEQ ID No1, a sequence complementary thereto, or a fragment thereof. Thenucleotide differences as regards to the nucleotide sequences of SEQ IDNo 1 are generally randomly distributed throughout the entire nucleicacid. Nevertheless, preferred nucleic acids are those wherein thedifferences as regards to the nucleotide sequences of SEQ ID No 1 arepredominantly located outside the coding sequences contained in theexons.

[0085] Another object of the invention consists of a purified, isolated,or recombinant nucleic acid that hybrizes with the sequence of SEQ ID No1 or a complementary sequence thereto or a variant thereof, under thestringent hybridization conditions as defined above.

[0086] These nucleic acids, as well as their fragments and variants, maybe used as oligonucleotide primers or probes in order to detect thepresence of a copy of the CIDE B gene in a test sample, or alternativelyin order to amplify a target nucleotide sequence within the CIDE Bsequences.

[0087] The CIDE B gene has 5 exons. The exon and intron positions in SEQID No 1 are detailed below in Table A. TABLE A Position in Position inSEQ ID No 1 SEQ ID NO 1 Exon Beginning End Intron Beginning End 1 28032922 1 2923 3224 2 3225 3369 2 3370 4216 3 4217 4366 3 4367 4602 4 46034793 4 4794 4974 55 4975 5555

[0088] Consequently, the invention also concerns a purified or isolatednucleic acid comprising a nucleotide sequence selected from the groupconsisting of the exons 1, 2, 3, 4, and 5 of the CIDE B gene, a sequencecomplementary thereto, a fragment or a variant thereof.

[0089] The invention also deals with a purified or isolated nucleic acidcomprising a combination of at least two polynucleotides selected fromthe group consisting of the exons 1, 2, 3, 4, and 5 of the CIDE B gene,wherein the polynucleotides are ordered within the nucleic acid, fromthe 5′ end to the 3′ end of said nucleic acid, in the same order than inthe SEQ ID No 1.

[0090] Thus, the invention embodies purified, isolated, or recombinantpolynucleotides comprising a nucleotide sequence selected from the groupconsisting of the introns of the CIDE B gene, or a sequencecomplementary thereto.

[0091] Particularly preferred nucleic acids of the invention includeisolated, purified, or recombinant polynucleotides comprising acontiguous span of at least 35, 40, 50, 60, 70, 80, 90, 100, 150, 200,500, or 1000 nucleotides of the nucleotide sequence of SEQ ID No 1, orthe complements thereof. Additionally preferred nucleic acids of theinvention include isolated, purified, or recombinant polynucleotidescomprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40,50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No1 or the complements thereof, wherein said contiguous span comprises atleast 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ IDNo 1: 1-1000, 1001-2000, 2001-3000, 3001-4000, 4001-5000, 5001-6000,6001-7000, 7001-8000, 8001-9000, 9001-10000, 10001-10961.

[0092] While this section is entitled “Genomic Sequences of CIDE B,” itshould be noted that nucleic acid fragments of any size and sequence mayalso be comprised by the polynucleotides described in this section,flanking the genomic sequences of CIDE B on either side or between twoor more such genomic sequences.

CIDE B cDNA Sequences

[0093] The inventors have discovered that the expression of the CIDE Bgene leads to the production of at least one mRNA molecule, the nucleicacid sequence of which is set forth in SEQ ID No 2.

[0094] Another object of the invention consists of a purified orisolated nucleic acid comprising the nucleotide sequence of SEQ ID No 2or fragments or variants thereof, or a complementary sequence thereto.

[0095] The invention also pertains to a purified or isolated nucleicacid having at least 70, 75, 80, 85, 90, or 95% nucleotide identity withthe nucleotide sequence of SEQ ID No 2, a sequence complementarythereto, or a fragment thereof.

[0096] Another object of the invention consists of a purified, isolated,or recombinant nucleic acid that hybridizes with the sequence of SEQ IDNo 2 or a complementary sequence thereto or a variant thereof, under thestringent hybridization conditions as defined above.

[0097] The nucleotide differences as regards to the nucleotide sequenceof SEQ ID No 2 are generally randomly distributed throughout the entirenucleic acid. Nevertheless, preferred nucleic acids are those whereinthe nucleotide differences as regards to the nucleotide sequence of SEQID No 2 are predominantly located outside the coding sequences, and moreprecisely in the 5′-UTR and the 3′-UTR sequences contained in thenucleotide sequence of SEQ ID No 2.

[0098] The cDNA of SEQ ID No 2 includes a 5′-UTR region starting fromthe nucleotide at position 1 and ending at the nucleotide in position 79of SEQ ID No 2. The cDNA of SEQ ID No 2 includes a 3 ′-UTR regionstarting from the nucleotide at position 740 and ending at thenucleotide at position 1187 of SEQ ID No 2.

[0099] Consequently, the invention concerns a purified or isolatednucleic acid comprising a nucleotide sequence selected from a groupconsisting of the 5′UTR and 3′UTR of the CIDE B cDNA, a sequencecomplementary thereto, a fragment or a variant thereof.

[0100] The middle line of FIG. 1 depicts the main structural features ofa purified or isolated nucleic acid consisting of a CIDE B cDNA. The5′-end sequence of this cDNA, more particularly the nucleotide sequencecomprised between the nucleotide in position 1 and the nucleotide inposition 247 of the nucleic acid of SEQ ID No 2 molecule corresponds tothe nucleotide sequence of a 5′-EST that has been obtained from a humanliver cDNA library. This 5′-EST is also part of the invention.

[0101] The invention also relates to isolated, purified, or recombinantpolynucleotides comprising a contiguous span of at least 35, 40, 50, 60,70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of the nucleotidesequence of SEQ ID No 2, or the complements thereof. Particularlypreferred nucleic acids of the invention include isolated, purified, orrecombinant polynucleotides comprising a contiguous span of at least 12,15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein saidcontiguous span comprises at least 1, 2, 3, 5, or 10 of the followingnucleotide positions of SEQ ID No 2: 1-78, 91-190, 208-229, 243-288,301-328, 364-394, 409-457, 478-490, 505-508, 529-597, 616-633,656-667,682-688,703-1188.

[0102] While this section is entitled “CIDE B cDNA Sequences,” it shouldbe noted that nucleic acid fragments of any size and sequence may alsobe comprised by the polynucleotides described in this section, flankingthe genomic sequences of CIDE B on either side or between two or moresuch genomic sequences.

Coding Regions of CIDE B

[0103] The CIDE B open reading frame is contained in the correspondingmRNA of SEQ ID No 2 and is a further object of the present invention.

[0104] More precisely, the effective CIDE B coding sequence (CDS) iscomprised between the nucleotide at position 80 (first nucleotide of theATG codon) and the nucleotide at position 739 (end nucleotide of the TAAcodon) of SEQ ID No 2. A purified or isolated polynucleotide comprisingthe CIDE B coding region defined above is another object of theinvention.

[0105] The present invention concerns a purified or isolated nucleicacid encoding a human CIDE B protein, wherein said CIDE B proteincomprises an amino acid sequence of SEQ ID No 3, a nucleotide sequencecomplementary thereto, a fragment or a variant thereof. The presentinvention also embodies isolated, purified, and recombinantpolynucleotides which encode a polypeptides comprising a contiguous spanof at least 35, 40, 50, or 100 amino acids of SEQ ID No 3. In apreferred embodiment, the present invention embodies isolated, purified,and recombinant polynucleotides which encode a polypeptides comprising acontiguous span of at least 8 amino acids, preferably at least 10 aminoacids, more preferably at least 12, 15, 20, 25, 30, 40, 50, 100, 150 or200 amino acids of SEQ ID No 3, wherein said contiguous span includes atleast 1, 2, 3, 5 or 10 of the following amino acid positions: 1-29,47-70, 103-115, 124, 134, 169-185, and 203-219. In an additionalpreferred embodiment, the present invention embodies isolated, purified,and recombinant polynucleotides which encode a polypeptides comprising acontiguous span of at least 6 amino acids, preferably at least 8 to 10amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, 100,150 or 200 amino acids of SEQ ID No 3, wherein said contiguous spanincludes at least 1, 2, 3, 5 or of the following amino acid positions:7-11, 18-29, 47, 55-63, 70, 103-104, 111-115, 124, 134, 169-173,181-185, and 203-219.

[0106] The above disclosed polynucleotide that contains the codingsequence of the CIDE B gene of the invention may be expressed in adesired host cell or a desired host organism, when this polynucleotideis placed under the control of suitable expression signals. Theexpression signals may be either the expression signals contained in theregulatory regions in the CIDE B gene of the invention or in contrast beexogenous regulatory nucleic sequences. Such a polynucleotide, whenplaced under the suitable expression signals, may also be inserted in avector for its expression.

CIDE B Regulatory Sequences

[0107] As already mentioned hereinbefore, the genomic sequence of theCIDE B gene contains regulatory sequences both in the non-coding5′-flanking region and in the non-coding 3′-flanking region that borderthe CIDE B coding region containing the exons of this gene.

[0108] The 5′-regulatory sequence of the CIDE B gene comprises thenucleotide sequence which is localized between the nucleotide inposition 1 and the nucleotide in position 2802 of the nucleotidesequence of SEQ ID No 1. This polynucleotide would contain the promotersite.

[0109] The 3′-regulatory sequence of the CIDE B gene comprises thenucleotide sequence which is localized between the nucleotide inposition 5556 and the nucleotide in position 10961 of the nucleotidesequence of SEQ ID No 1.

[0110] Polynucleotides derived from the CIDE B regulatory regionsdescribed above are useful in order to detect the presence of at least acopy of a nucleotide sequence of SEQ ID No 1, or a fragment or a variantthereof in a test sample.

[0111] Thus, the present invention also concerns a purified or isolatednucleic acid comprising a polynucleotide which is selected from thegroup consisting of the 5′ and 3′ regulatory regions, or a sequencecomplementary thereto or a biologically active fragment or variantthereof. “5′ regulatory region” refers to the nucleotide sequencelocated between positions 1 and 2802 of SEQ ID No 1. “3′ regulatoryregion” refers to the nucleotide sequence located between positions 5556and 10961 of SEQ ID No 1.

[0112] The invention also pertains to a purified or isolated nucleicacid comprising a polynucleotide having at least 70, 75, 80, 85, 90, or95% nucleotide identity with the nucleotide sequence selected from thegroup consisting of the 5′ and 3′ regulatory regions, or a sequencecomplementary thereto or a biologically active fragment thereof.

[0113] Another object of the invention consists of purified, isolated orrecombinant nucleic acids comprising a polynucleotide that hybridizes,under the stringent hybridization conditions defined herein, with apolynucleotide selected from the group consisting of the nucleotidesequences of the 5′- and 3′ regulatory regions, or a sequencecomplementary thereto or a variant thereof or a biologically activefragment thereof

[0114] The promoter activity of the regulatory regions contained in the5′ regulatory region of CIDE B can be assessed as described below.

[0115] In order to identify the relevant biologically activepolynucleotide fragments or variants of the 5′ and 3′ regulatoryregions, the one skill in the art will refer to the book of Sambrook etal. (1989) which describes the use of a recombinant vector carrying amarker gene (i.e. beta galactosidase, chloramphenicol acetyltransferase, etc.) the expression of which will be detected when placedunder the control of a biologically active polynucleotide fragments orvariants of the 5′ and 3′ regulatory regions. Genomnic sequences locatedupstream of the first exon of the CIDE B gene are cloned into a suitablepromoter reporter vector, such as the pSEAP-Basic, pSEAP-Enhancer,pβgal-Basic, pβgal-Enhancer, or pEGFP-1 Promoter Reporter vectorsavailable from Clontech, or pGL2-basic or pGL3-basic promoterlessluciferase reporter gene vector from Promega. Briefly, each of thesepromoter reporter vectors include multiple cloning sites positionedupstream of a reporter gene encoding a readily assayable protein such assecreted alkaline phosphatase, luciferase, beta galactosidase, or greenfluorescent protein. The sequences upstream the CIDE B coding region areinserted into the cloning sites upstream of the reporter gene in bothorientations and introduced into an appropriate host cell. The level ofreporter protein is assayed and compared to the level obtained from avector which lacks an insert in the cloning site. The presence of anelevated expression level in the vector containing the insert withrespect to the control vector indicates the presence of a promoter inthe insert. If necessary, the upstream sequences can be cloned intovectors which contain an enhancer for increasing transcription levelsfrom weak promoter sequences. A significant level of expression abovethat observed with the vector lacking an insert indicates that apromoter sequence is present in the inserted upstream sequence.

[0116] Promoter sequences within the upstream genomic DNA may be furtherdefined by constructing nested 5′ and/or 3′ deletions in the upstreamDNA using conventional techniques such as Exonuclease III or appropriaterestriction endonuclease digestion. The resulting deletion fragments canbe inserted into the promoter reporter vector to determine whether thedeletion has reduced or obliterated promoter activity, such asdescribed, for example, by Coles et al. (1998). In this way, theboundaries of the promoters may be defined. If desired, potentialindividual regulatory sites within the promoter may be identified usingsite directed mutagenesis or linker scanning to obliterate potentialtranscription factor binding sites within the promoter individually orin combination. The effects of these mutations on transcription levelsmay be determined by inserting the mutations into cloning sites inpromoter reporter vectors. This type of assay is well-known to thoseskilled in the art and is described in WO 97/17359, U.S. Pat. No.5,374,544, EP 582 796, U.S. Pat. No. 5,698,389, U.S. Pat. No. 5,643,746,U.S. Pat. No. 5,502,176, and U.S. Pat. No. 5,266,488.

[0117] The strength and the specificity of the promoter of the CIDE Bgene can be assessed through the expression levels of a detectablepolynucleotide operably linked to the CIDE B promoter in different typesof cells and tissues. The detectable polynucleotide may be either apolynucleotide that specifically hybridizes with a predefinedoligonucleotide probe, or a polynucleotide encoding a detectableprotein, including a CIDE B polypeptide or a fragment or a variantthereof. This type of assay is well-known to those skilled in the artand is described in U.S. Pat. No. 5,502,176, and U.S. Pat. No.5,266,488.

[0118] Polynucleotides carrying the 5′ and 3′ regulatory regions of CIDEB coding region may be advantageously used to control thetranscriptional and translational activity of an heterologouspolynucleotide of interest.

[0119] Thus, the present invention also concerns a purified or isolatednucleic acid comprising a polynucleotide which is selected from thegroup consisting of the 5′ and 3′ regulatory regions of CIDE B, or asequence complementary thereto or a biologically active fragment orvariant thereof.

[0120] Preferred fragments of the 5′ regulatory region of CIDE B have alength of about 1000 nucleotides, more particularly about 500nucleotides, more preferably 200 nucleotides and most preferably about100 nucleotides.

[0121] Preferred fragments of 3′ regulatory region of CIDE B have alength of about 1000 nucleotides, more particularly about 500nucleotides, more preferably 200 nucleotides and most preferably about100 nucleotides.

[0122] By a “biologically active” polynucleotide derivative of the 5′and 3′ regulatory regions of the CIDE B is intended a polynucleotidecomprising or alternatively consisting in a fragment of saidpolynucleotide which is functional as a regulatory region for expressinga recombinant polypeptide or a recombinant polynucleotide in arecombinant cell host.

[0123] For the purpose of the invention, a nucleic acid orpolynucleotide is “functional” as a regulatory region for expressing arecombinant polypeptide or a recombinant polynucleotide if saidregulatory polynucleotide contains nucleotide sequences which containtranscriptional and translational regulatory information, and suchsequences are “operably linked” to nucleotide sequences which encode thedesired polypeptide or the desired polynucleotide.

[0124] These regulatory polynucleotides can also be prepared by nucleicacid chemical synthesis, as described elsewhere in the specification,where oligonucleotide probes or primers synthesis is disclosed.

[0125] The regulatory polynucleotides according to the invention may beadvantageously part of a recombinant expression vector that may be usedto express a coding sequence in a desired host cell or host organism.The recombinant expression vectors according to the invention aredescribed elsewhere in the specification.

[0126] A preferred 5′-regulatory polynucleotide of the inventionincludes the 5′-UTR of CIDE B, or a biologically active fragment orvariant thereof.

[0127] A preferred 3′-regulatory polynucleotide of the inventionincludes a 3′-UTR of CIDE B, or a biologically active fragment orvariant thereof. This preferred 3′-regulatory polynucleotide carries apolyadenylation site located between the nucleotide in position 1158 andthe nucleotide in position 1163 of the nucleic acid of SEQ ID No 2.

[0128] The regulatory polynucleotides of the invention may be preparedfrom any of the nucleotide sequence of SEQ ID No 1 by cleavage usingsuitable restriction enzymes, as described for example in the book ofSambrook et al. (1989). The regulatory polynucleotides may also beprepared by digestion of any of SEQ ID No 1 by an exonuclease enzyme,such as for example Bal31 (Wabiko et al., 1986).

[0129] A further object of the invention consists of a purified orisolated nucleic acid comprising:

[0130] a) a nucleic acid comprising the 5′ regulatory region of CIDE Bor a biologically active fragment or variant thereof,

[0131] b) a polynucleotide encoding a desired polypeptide or nucleicacid operably linked to said 5′ regulatory polynucleotide or itsbiologically active fragment or variant thereof;

[0132] c) optionally, a nucleic acid comprising the 3′ regulatory regionof CIDE B or a biologically active fragment or variant thereof

[0133] In a specific embodiment of the nucleic acid defined above, saidnucleic acid includes the 5′-UTR of CIDE B, or a biologically activefragment or variant thereof. In a second specific embodiment of thenucleic acid defined above, said nucleic acid includes the 3′-UTR ofCIDE B, or a biologically active fragment or variant thereof.

[0134] The 5′ regulatory region of CIDE B, or its biologically activefragments or variants, is advantageously operably linked at the 5′-endof the polynucleotide encoding the desired polypeptide orpolynucleotide.

[0135] The 3′ regulatory region of CIDE B, or its biologically activefragments and variants, is advantageously placed at the 3′-end of thepolynucleotide encoding the desired polypeptide or polynucleotide.

[0136] The desired polypeptide encoded by the above described nucleicacid may be of various nature or origin, encompassing proteins ofprokaryotic or eukaryotic origin. Among the polypeptides expressed underthe control of a CIDE B regulatory region, there may be cited bacterial,fungal or viral antigens. Also encompassed are eukaryotic proteins suchas intracellular proteins, like “house keeping” proteins, membrane-boundproteins, like receptors, and secreted proteins like the numerousendogenous mediators such as cytokines. The desired polypeptide may bethe CIDE B protein, especially the protein of the amino acid sequence ofSEQ ID No 3, or a fragment or a variant thereof.

[0137] The desired nucleic acids encoded by the above describedpolynucleotide, usually a RNA molecule, may be complementary to adesired coding polynucleotide, for example to the CIDE B codingsequence, and thus useful as an antisense polynucleotide.

[0138] Such a polynucleotide may be included in a recombinant expressionvector in order to express the desired polypeptide or the desirednucleic acid in host cell or in a host organism. Suitable recombinantvectors that contain a polynucleotide such as described hereinbefore aredisclosed elsewhere in the specification.

Oligonucleotide Probes and Primers

[0139] Polynucleotides derived from the CIDE B gene are useful in orderto detect the presence of at least a copy of a nucleotide sequence ofSEQ ID No 1 or 2, or a fragment, complement, or variant thereof in atest sample.

[0140] Particularly preferred probes and primers of the inventioninclude isolated, purified, or recombinant polynucleotides comprising acontiguous span of at least 35, 40, 50, 60, 70, 80, 90, 100, 150, 200,500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof.Further preferred probes and primers of the invention include isolated,purified, or recombinant polynucleotides comprising a contiguous span ofat least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof,wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of thefollowing nucleotide positions of SEQ ID No 1: 1-1000, 1001-2000,2001-3000, 3001-4000, 4001-5000, 5001-6000, 6001-7000, 7001-8000,8001-9000, 9001-10000, 10001-10961.

[0141] Other preferred probes and primers of the invention includeisolated, purified, or recombinant polynucleotides comprising acontiguous span of at least 35, 40, 50, 60, 70, 80, 90, 100, 150, 200,500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof.Additional preferred probes and primers of the invention includeisolated, purified, or recombinant polynucleotides comprising acontiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70,80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or thecomplements thereof, wherein said contiguous span comprises at least 1,2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 2:1-78, 91-190, 208-229, 243-288, 301-328, 364-394, 409-457, 478-490,505-508, 529-597, 616-633, 656-667, 682-688, 703-1188.

[0142] Thus, the invention also relates to nucleic acid probescharacterized in that they hybridize specifically, under the stringenthybridization conditions defined above, with a nucleic acid selectedfrom the group consisting of the nucleotide sequences of SEQ ID Nos 1and 2 or a variant thereof or a sequence complementary thereto.

[0143] In one embodiment the invention encompasses isolated, purified,and recombinant polynucleotides consisting of, or consisting essentiallyof a contiguous span of 8 to 50 nucleotides of any one of SEQ ID No 1and the complement thereof, wherein said span includes a CIDE B -relatedbiallelic marker in said sequence; optionally, wherein said CIDE B-related biallelic marker is selected from the group consisting of thebiallelic markers 12-73-49 and 12-74-38, and the complements thereof;optionally, wherein said contiguous span is 18 to 47 nucleotides inlength and said biallelic marker is within 4 nucleotides of the centerof said polynucleotide; optionally, wherein said polynucleotide consistsof said contiguous span and said contiguous span is 25 nucleotides inlength and said biallelic marker is at the center of saidpolynucleotide; optionally, wherein the 3′ end of said contiguous spanis present at the 3′ end of said polynucleotide; and optionally, whereinthe 3′ end of said contiguous span is located at the 3′ end of saidpolynucleotide and said biallelic marker is present at the 3′ end ofsaid polynucleotide. In a preferred embodiment, said probes comprises,consists of, or consists essentially of a sequence selected from thefollowing sequences: P(12-73-49) and P(12-74-38) and the complementarysequences thereto.

[0144] In another embodiment the invention encompasses isolated,purified and recombinant polynucleotides comprising, consisting of, orconsisting essentially of a contiguous span of 8 to 50 nucleotides ofSEQ ID No 1, or the complements thereof, wherein the 3′ end of saidcontiguous span is located at the 3′ end of said polynucleotide, andwherein the 3′ end of said polynucleotide is located within 20nucleotides upstream of a CIDE B -related biallelic marker in saidsequence; optionally, wherein said CIDE B -related biallelic marker isselected from the group consisting of the biallelic markers 12-73-49 and12-74-38, and the complements thereof; optionally, wherein the 3′ end ofsaid polynucleotide is located 1 nucleotide upstream of said CIDE B-related biallelic marker in said sequence; and optionally, wherein saidpolynucleotide consists essentially of a sequence selected from thefollowing sequences: D(12-73-49), D(12-74-38), E(12-73-49), andE(12-74-38).

[0145] In a further embodiment, the invention encompasses isolated,purified, or recombinant polynucleotides comprising, consisting of, orconsisting essentially of a sequence selected from the followingsequences: B(12-73), B(12-74), C(12-73), and C(12-74).

[0146] In an additional embodiment, the invention encompassespolynucleotides for use in hybridization assays, sequencing assays, andenzyme-based mismatch detection assays for determining the identity ofthe nucleotide at a CIDE B -related biallelic marker in SEQ ID No 1 orthe complements thereof, as well as polynucleotides for use inamplifying segments of nucleotides comprising a CIDE B -relatedbiallelic marker in SEQ ID No 1 or the complements thereof; optionally,wherein said CIDE B -related biallelic marker is selected from the groupconsisting of the biallelic markers 12-73-49 and 12-74-38, and thecomplements thereof.

[0147] A probe or a primer according to the invention has between 8 and1000 nucleotides in length, or is specified to be at least 12, 15, 18,20, 25, 35, 40, 50, 60, 70, 80, 100, 250, 500 or 1000 nucleotides inlength. More particularly, the length of these probes and primers canrange from 8, 10, 15, 20, or 30 to 100 nucleotides, preferably from 10to 50, more preferably from 15 to 30 nucleotides. Shorter probes andprimers tend to lack specificity for a target nucleic acid sequence andgenerally require cooler temperatures to form sufficiently stable hybridcomplexes with the template. Longer probes and primers are expensive toproduce and can sometimes self-hybridize to form hairpin structures. Theappropriate length for primers and probes under a particular set ofassay conditions may be empirically determined by one of skill in theart. A preferred probe or primer consists of a nucleic acid comprising apolynucleotide selected from the group of the nucleotide sequences ofP(12-73-49) and P(12-74-38) and the complementary sequence thereto,B(12-73), B(12-74), C(12-73), C(12-74), D(12-73-49), D(12-74-38), forwhich the respective locations in the sequence listing are provided inTables 1, 2, 3 and 4.

[0148] The formation of stable hybrids depends on the meltingtemperature (Tm) of the DNA. The Tm depends on the length of the primeror probe, the ionic strength of the solution and the G+C content. Thehigher the G+C content of the primer or probe, the higher is the meltingtemperature because G:C pairs are held by three H bonds whereas A:Tpairs have only two. The GC content in the probes of the inventionusually ranges between 10 and 75%, preferably between 35 and 60%, andmore preferably between 40 and 55%.

[0149] The primers and probes can be prepared by any suitable method,including, for example, cloning and restriction of appropriate sequencesand direct chemical synthesis by a method such as the phosphodiestermethod of Narang et al.(1979), the phosphodiester method of Brown eta.(1979), the diethylphosphoramidite method of Beaucage et al.(1981) andthe solid support method described in EP 0 707 592.

[0150] Detection probes are generally nucleic acid sequences oruncharged nucleic acid analogs such as, for example peptide nucleicacids which are disclosed in International Patent application WO92/20702, morpholino analogs which are described in U.S. Pat. Nos.5,185,444; 5,034,506 and 5,142,047. The probe may have to be rendered“non-extendable” in that additional dNTPs cannot be added to the probe.In and of themselves analogs usually are non-extendable and nucleic acidprobes can be rendered non-extendable by modifying the 3′ end of theprobe such that the hydroxyl group is no longer capable of participatingin elongation. For example, the 3′ end of the probe can befunctionalized with the capture or detection label to thereby consume orotherwise block the hydroxyl group. Alternatively, the 3′ hydroxyl groupsimply can be cleaved, replaced or modified, U.S. patent applicationSer. No. 07/049,061 filed Apr. 19, 1993 describes modifications, whichcan be used to render a probe non-extendable.

[0151] Any of the polynucleotides of the present invention can belabeled, if desired, by incorporating any label known in the art to bedetectable by spectroscopic, photochemical, biochemical,immumunochemical, or chemical means. For example, useful labels includeradioactive substances (including, ³²p, ³⁵S, ³H, ¹²⁵I), fluorescent dyes(including, 5-bromodesoxyuridin, fluorescein, acetylaminofluorene,digoxigenin) or biotin. Preferably, polynucleotides are labeled at their3′ and 5′ ends. Examples of non-radioactive labeling of nucleic acidfragments are described in the French patent No. FR-7810975 or by Urdeaet al (1988) or Sanchez-Pescador et al (1988). In addition, the probesaccording to the present invention may have structural characteristicssuch that they allow the signal amplification, such structuralcharacteristics being, for example, branched DNA probes as thosedescribed by Urdea et al. in 1991 or in the European patent No. EP 0 225807 (Chiron).

[0152] A label can also be used to capture the primer, so as tofacilitate the immobilization of either the primer or a primer extensionproduct, such as amplified DNA, on a solid support. A capture label isattached to the primers or probes and can be a specific binding memberwhich forms a binding pair with the solid's phase reagent's specificbinding member (e.g. biotin and streptavidin). Therefore depending uponthe type of label carried by a polynucleotide or a probe, it may beemployed to capture or to detect the target DNA. Further, it will beunderstood that the polynucleotides, primers or probes provided herein,may, themselves, serve as the capture label. For example, in the casewhere a solid phase reagent's binding member is a nucleic acid sequence,it may be selected such that it binds a complementary portion of aprimer or probe to thereby immobilize the primer or probe to the solidphase. In cases where a polynucleotide probe itself serves as thebinding member, those skilled in the art will recognize that the probewill contain a sequence or “tail” that is not complementary to thetarget. In the case where a polynucleotide primer itself serves as thecapture label, at least a portion of the primer will be free tohybridize with a nucleic acid on a solid phase. DNA Labeling techniquesare well known to the skilled technician.

[0153] The probes of the present invention are useful for a number ofpurposes. They can be notably used in Southern hybridization to genomicDNA. The probes can also be used to detect PCR amplification products.They may also be used to detect mismatches in the CIDE B gene or mRNAusing other techniques.

[0154] Any of the polynucleotides, primers and probes of the presentinvention can be conveniently immobilized on a solid support. Solidsupports are known to those skilled in the art and include the walls ofwells of a reaction tray, test tubes, polystyrene beads, magnetic beads,nitrocellulose strips, membranes, microparticles such as latexparticles, sheep (or other animal) red blood cells, duracytes andothers. The solid support is not critical and can be selected by oneskilled in the art. Thus, latex particles, microparticles, magnetic ornon-magnetic beads, membranes, plastic tubes, walls of microtiter wells,glass or silicon chips, sheep (or other suitable animal's) red bloodcells and duracytes are all suitable examples. Suitable methods forimmobilizing nucleic acids on solid phases include ionic, hydrophobic,covalent interactions and the like. A solid support, as used herein,refers to any material which is insoluble, or can be made insoluble by asubsequent reaction. The solid support can be chosen for its intrinsicability to attract and immobilize the capture reagent. Alternatively,the solid phase can retain an additional receptor which has the abilityto attract and immobilize the capture reagent. The additional receptorcan include a charged substance that is oppositely charged with respectto the capture reagent itself or to a charged substance conjugated tothe capture reagent. As yet another alternative, the receptor moleculecan be any specific binding member which is immobilized upon (attachedto) the solid support and which has the ability to immobilize thecapture reagent through a specific binding reaction. The receptormolecule enables the indirect binding of the capture reagent to a solidsupport material before the performance of the assay or during theperformance of the assay. The solid phase thus can be a plastic,derivatized plastic, magnetic or non-magnetic metal, glass or siliconsurface of a test tube, microliter well, sheet, bead, microparticle,chip, sheep (or other suitable animal's) red blood cells, duracytes® andother configurations known to those of ordinary skill in the art. Thepolynucleotides of the invention can be attached to or immobilized on asolid support individually or in groups of at least 2, 5, 8, 10, 12, 15,20, or 25 distinct polynucleotides of the invention to a single solidsupport. In addition, polynucleotides other than those of the inventionmay be attached to the same solid support as one or more polynucleotidesof the invention.

[0155] Consequently, the invention also comprises a method for detectingthe presence of a nucleic acid comprising a nucleotide sequence selectedfrom a group consisting of SEQ ID Nos 1 and 2, a fragment or a variantthereof and a complementary sequence thereto in a sample, said methodcomprising the following steps of:

[0156] a) bringing into contact a nucleic acid probe or a plurality ofnucleic acid probes which can hybridize with a nucleotide sequenceincluded in a nucleic acid selected form the group consisting of thenucleotide sequences of SEQ ID Nos 1 and 2, a fragment or a variantthereof and a complementary sequence thereto and the sample to beassayed; and

[0157] b) detecting the hybrid complex formed between the probe and anucleic acid in the sample.

[0158] The invention further concerns a kit for detecting the presenceof a nucleic acid comprising a nucleotide sequence selected from a groupconsisting of SEQ ID Nos 1 and 2, a fragment or a variant thereof and acomplementary sequence thereto in a sample, said kit comprising:

[0159] a) a nucleic acid probe or a plurality of nucleic acid probeswhich can hybridize with a nucleotide sequence included in a nucleicacid selected form the group consisting of the nucleotide sequences ofSEQ ID Nos 1 and 2, a fragment or a variant thereof and a complementarysequence thereto; and

[0160] b) optionally, the reagents necessary for performing thehybridization reaction.

[0161] In a first preferred embodiment of this detection method and kit,said nucleic acid probe or the plurality of nucleic acid probes arelabeled with a detectable molecule. In a second preferred embodiment ofsaid method and kit, said nucleic acid probe or the plurality of nucleicacid probes has been immobilized on a substrate. In a third preferredembodiment, the nucleic acid probe or the plurality of nucleic acidprobes comprise either a sequence which is selected from the groupconsisting of the nucleotide sequences of of P(12-73-49) and P(12-74-38)and the complementary sequence thereto, B(12-73), B(12-74), C(12-73),C(12-74), D(12-73-49), D(12-74-38), E(12-73-49), and E(12-74-38) or abiallelic marker selected from the group consisting of the biallelicmarkers 12-73-49 and 12-74-38 and the complements thereto.

[0162] Oligonucleotide Arrays

[0163] A substrate comprising a plurality of oligonucleotide primers orprobes of the invention may be used either for detecting or amplifyingtargeted sequences in the CIDE B gene and may also be used for detectingmutations in the coding or in the non-coding sequences of the CIDE Bgene.

[0164] Any polynucleotide provided herein may be attached in overlappingareas or at random locations on the solid support. Alternatively thepolynucleotides of the invention may be attached in an ordered arraywherein each polynucleotide is attached to a distinct region of thesolid support which does not overlap with the attachment site of anyother polynucleotide. Preferably, such an ordered array ofpolynucleotides is designed to be “addressable” where the distinctlocations are recorded and can be accessed as part of an assayprocedure. Addressable polynucleotide arrays typically comprise aplurality of different oligonucleotide probes that are coupled to asurface of a substrate in different known locations. The knowledge ofthe precise location of each polynucleotides location makes these“addressable” arrays particularly useful in hybridization assays. Anyaddressable array technology known in the art can be employed with thepolynucleotides of the invention. One particular embodiment of thesepolynucleotide arrays is known as the Genechips™, and has been generallydescribed in U.S. Pat. No. 5,143,854; PCT publications WO 90/15070 and92/10092. These arrays may generally be produced using mechanicalsynthesis methods or light directed synthesis methods which incorporatea combination of photolithographic methods and solid phaseoligonucleotide synthesis (Fodor et al., 1991). The immobilization ofarrays of oligonucleotides on solid supports has been rendered possibleby the development of a technology generally identified as “Very LargeScale Immobilized Polymer Synthesis” (VLSIPS™) in which, typically,probes are immobilized in a high density array on a solid surface of achip. Examples of VLSIPS™ technologies are provided in U.S. Pat. Nos.5,143,854; and 5,412,087 and in PCT Publications WO 90/15070, WO92/10092 and WO 95/11995, which describe methods for formingoligonucleotide arrays through techniques such as light-directedsynthesis techniques. In designing strategies aimed at providing arraysof nucleotides immobilized on solid supports, further presentationstrategies were developed to order and display the oligonucleotidearrays on the chips in an attempt to maximize hybridization patterns andsequence information. Examples of such presentation strategies aredisclosed in PCT Publications WO 94/12305, WO 94/11530, WO 97/29212 andWO 97/31256.

[0165] In another embodiment of the oligonucleotide arrays of theinvention, an oligonucleotide probe matrix may advantageously be used todetect mutations occurring in the CIDE B gene and preferably in itsregulatory region. For this particular purpose, probes are specificallydesigned to have a nucleotide sequence allowing their hybridization tothe genes that carry known mutations (either by deletion, insertion orsubstitution of one or several nucleotides). By known mutations, it ismeant, mutations on the CIDE B gene that have been identified according,for example to the technique used by Huang et al.(1996) or Samson etal.(1996).

[0166] Another technique that is used to detect mutations in the CIDE Bgene is the use of a high-density DNA array. Each oligonucleotide probeconstituting a unit element of the high density DNA array is designed tomatch a specific subsequence of the CIDE B genomic DNA or cDNA. Thus, anarray consisting of oligonucleotides complementary to subsequences ofthe target gene sequence is used to determine the identity of the targetsequence with the wild gene sequence, measure its amount, and detectdifferences between the target sequence and the reference wild genesequence of the CIDE B gene. In one such design, termed 4L tiled array,is implemented a set of four probes (A, C, G, T), preferably15-nucleotide oligomers. In each set of four probes, the perfectcomplement will hybridize more strongly than mismatched probes.Consequently, a nucleic acid target of length L is scanned for mutationswith a tiled array containing 4L probes, the whole probe set containingall the possible mutations in the known wild reference sequence. Thehybridization signals of the 15-mer probe set tiled array are perturbedby a single base change in the target sequence. As a consequence, thereis a characteristic loss of signal or a “footprint” for the probesflanking a mutation position. This technique was described by Chee etal. in 1996.

[0167] Consequently, the invention concerns an array of nucleic acidmolecules comprising at least one polynucleotide described above asprobes and primers. Preferably, the invention concerns an array ofnucleic acid comprising at least two polynucleotides described above asprobes and primers.

[0168] A further object of the invention consists of an array of nucleicacid sequences comprising either at least one of the sequences selectedfrom the group consisting of P(12-73-49), P(12-74-38), B(12-73),B(12-74), C(12-73), C(12-74), D(12-73-49), and E(12-74-38), thesequences complementary thereto, a fragment thereof of at least 8, 10,12, 15, 18, 20, 25, 30, or 40 consecutive nucleotides thereof, and atleast one sequence comprising a biallelic marker selected from the groupconsisting of the biallelic markers 12-73-49 and 12-74-38 and thecomplements thereto.

[0169] The invention also pertains to an array of nucleic acid sequencescomprising either at least two of the sequences selected from the groupconsisting of P(12-73-49), P(12-74-38), B(12-73), B(12-74), C(12-73),C(12-74), D(12-73-49), D(12-74-38), E(12-73-49), P(12-74-38), thesequences complementary thereto, a fragment thereof of at least 8consecutive nucleotides thereof, and at least two sequences comprising abiallelic marker selected from the group consisting of the biallelicmarkers 12-73-49 and 12-74-38 and the complements thereof.

Amplification of the CIDE B Gene

[0170] DNA Extraction

[0171] As for the source of the genomic DNA to be subjected to analysis,any test sample can be foreseen without any particular limitation. Thesetest samples include biological samples which can be tested by themethods of the present invention described herein and include human andanimal body fluids such as whole blood, serum, plasma, cerebrospinalfluid, urine, lymph fluids, and various external secretions of therespiratory, intestinal and genitourinary tracts, tears, saliva, milk,white blood cells, myelomas and the like; biological fluids such as cellculture supernatants; fixed tissue specimens including tumor andnon-tumor tissue and lymph node tissues; bone marrow aspirates and fixedcell specimens. The preferred source of genomic DNA used in the contextof the present invention is from peripheral venous blood of each donor.

[0172] The techniques of DNA extraction are well-known to the skilledtechnician. Such techniques are described notably by Lin et al. (1998)and by Mackey et al. (1998).

[0173] DNA Amplification

[0174] Amplification techniques that can be used in the context of thepresent invention include, but are not limited to, the ligase chainreaction (LCR) described in EP-A- 320 308, WO 9320227 and EP-A-439 182,the polymerase chain reaction (PCR, RT-PCR) and techniques such as thenucleic acid sequence based amplification (NASBA) described in GuatelliJ. C., et al.(1990) and in Compton J.(1991), Q-beta amplification asdescribed in European Patent Application No 4544610, strand displacementamplification as described in Walker et al.(1996) and EP A 684 315 and,target mediated amplification as described in PCT Publication WO9322461.

[0175] LCR and Gap LCR are exponential amplification techniques, bothdepend on DNA ligase to join adjacent primers annealed to a DNAmolecule. In Ligase Chain Reaction (LCR), probe pairs are used whichinclude two primary (first and second) and two secondary (third andfourth) probes, all of which are employed in molar excess to target. Thefirst probe hybridizes to a first segment of the target strand and thesecond probe hybridizes to a second segment of the target strand, thefirst and second segments being contiguous so that the primary probesabut one another in 5′ phosphate-3′hydroxyl relationship, and so that aligase can covalently fuse or ligate the two probes into a fusedproduct. In addition, a third (secondary) probe can hybridize to aportion of the first probe and a fourth (secondary) probe can hybridizeto a portion of the second probe in a similar abutting fashion. Ofcourse, if the target is initially double stranded, the secondary probesalso will hybridize to the target complement in the first instance. Oncethe ligated strand of primary probes is separated from the targetstrand, it will hybridize with the third and fourth probes, which can beligated to form a complementary, secondary ligated product. It isimportant to realize that the ligated products are functionallyequivalent to either the target or its complement. By repeated cycles ofhybridization and ligation, amplification of the target sequence isachieved. A method for multiplex LCR has also been described (WO9320227). Gap LCR (GLCR) is a version of LCR where the probes are notadjacent but are separated by 2 to 3 bases.

[0176] For amplification of mRNAs, it is within the scope of the presentinvention to reverse transcribe mRNA into cDNA followed by polymerasechain reaction (RT-PCR); or, to use a single enzyme for both steps asdescribed in U.S. Pat. No. 5,322,770 or, to use Asymmetric Gap LCR(RT-AGLCR) as described by Marshall et al.(1994). AGLCR is amodification of GLCR that allows the amplification of RNA.

[0177] The PCR technology is the preferred amplification technique usedin the present invention. A variety of PCR techniques are familiar tothose skilled in the art. For a review of PCR technology, see White(1997) and the publication entitled “PCR Methods and Applications”(1991, Cold Spring Harbor Laboratory Press). In each of these PCRprocedures, PCR primers on either side of the nucleic acid sequences tobe amplified are added to a suitably prepared nucleic acid sample alongwith dNTPs and a thermostable polymerase such as Taq polymerase, Pfupolymerase, or Vent polymerase. The nucleic acid in the sample isdenatured and the PCR primers are specifically hybridized tocomplementary nucleic acid sequences in the sample. The hybridizedprimers are extended. Thereafter, another cycle of denaturation,hybridization, and extension is initiated. The cycles are repeatedmultiple times to produce an amplified fragment containing the nucleicacid sequence between the primer sites. PCR has further been describedin several patents including U.S. Pat. Nos. 4,683,195; 4,683,202; and4,965,188.

[0178] The present invention also relates to a method for theamplification of a human CIDE B gene sequence, particularly of a portionof the genomic sequence of SEQ ID No 1 or of the cDNA sequence of SEQ IDNo 2, or a variant thereof in a test sample, said method comprising thesteps of:

[0179] a) contacting a test sample suspected of containing the targetedCIDE B gene sequence comprised in a nucleotide sequence selected from agroup consisting of SEQ ID Nos 1 and 2, or fragments or variants thereofwith amplification reaction reagents comprising a pair of amplificationprimers as described above and located on either side of thepolynucleotide region to be amplified, and

[0180] b) optionally, detecting the amplification products.

[0181] The invention also concerns a kit for the amplification of ahuman CIDE B gene sequence, particularly of a portion of the genomicsequence of SEQ ID No 1 or of the cDNA sequence of SEQ ID No 2, or avariant thereof in a test sample, wherein said kit comprises:

[0182] a) a pair of oligonucleotide primers located on either side ofthe CIDE B region to be amplified;

[0183] b) Optionally, the reagents necessary for performing theamplification reaction.

[0184] In one specific embodiment of the above amplification method andkit, the amplification primers are selected from the group consisting ofthe polynucleotides B(12-73), B(12-74), C(12-73), C(12-74), D(12-73-49),D(12-74-38), E(12-73-49), and #(12-74-38).

[0185] In another embodiment of the above amplification method and kit,the amplification product is detected by hybridization with a labeledprobe having a sequence which is complementary to the amplified region.

CIDE B Polypeptide and Peptide Fragments thereof

[0186] It is now easy to produce proteins in hight amounts by geneticengineering techniques through expression vectors such as plasmids,phages or phagemids. The polynucleotide that code for one thepolypeptides of the present invention is inserted in an appropriateexpression vector in order to produce the polypeptide of interestinvtro.

[0187] Thus, the present invention also concerns a method for producingone of the polypeptides described herein, and especially a polypeptideof SEQ ID No 3 or a fragment or a variant thereof, wherein said methodcomprises the steps of:

[0188] a) culturing, in an appropriate culture medium, a cell hostpreviously transformed or transfected with the recombinant vectorcomprising a nucleic acid encoding a CIDE B polypeptide, or a fragmentor a variant thereof;

[0189] b) harvesting the culture medium thus conditioned or lyse thecell host, for example by sonaication or by an osmotic shock;

[0190] c) separating or purifying, from the said culture medium, or fromthe pellet of the resultant host cell lysate the thus producedpolypeptide of interest.

[0191] d) Optionally characterizing the produce polypeptide of interest.

[0192] In a specific embodiment of the above method, step a) is precededby a step wherein the nucleic acid coding for a CIDE B polypeptide, or afragment or a variant thereof, is inserted in an appropriate vector,optionally afgter an appropriate cleavage of this amplified nucleic acidwith one or several restriction endonucleases. The nucleic acid codingfor a CIDE B polypeptide or a fragment or a variant thereof may be theresulting product of an amplification reaction using a pair of primersaccording to the invention (by SDA, TAS, 3SR, NASBA, TMA etc.).

[0193] The polypeptides according to the invention may be characterizedby binding onto an immounaffinity chromatography column on whichpolyclonal or monoclonal antibodies directed to a polypeptide of SEQ IDNo 3, or a fragment or a variant thereof, have previously beenimmobilized.

[0194] Purification of the recombinant proteins or peptides according tothe present invention may be carried out by passage onto a Nickel orCupper affinity chromatography column. The Nickel chromatography columnmay contain the Ni-NTA resin (Porath et al., 1975).

[0195] The polypeptides or peptides thus obtained may be purified, forexample by high performance liquid chromatography, such as reverse phaseand/or cationic exchange HPLC, as described by Rougeo et al. (1994). Thereason to prefer this kind of peptide or protein purification is thelack of byproducts found in the elution samples which renders theresultant purified protein or peptide more suitable for a therapeuticuse.

[0196] In a preferred embodiment, the CIDE B polypeptide comprises anamino acid sequence of SEQ ID No 3 or a fragment or a variant thereof.

[0197] The CIDE B polypeptide of the amino acid sequence of SEQ ID No 3has 219 amino acids in length.

[0198] The human CIDE B protein presents 85% of identity with the murineCIDE B. This level of identity shows that the protein of the presentinvention is the human homologue of the murine CIDE B. In contrast, thehuman CIDE B protein of the invention presents only 42% of identity withthe human CIDE A protein.

[0199] The invention also encompasses a purified, isolated, orrecombinant polypeptides comprising an amino acid sequence having atleast 90, 95, 98 or 99% amino acid identity with the amino acid sequenceof SEQ ID No 3 or a fragment thereof.

[0200] In a preferred embodiment, the CIDE B polypeptide comprises anamino acid sequence of SEQ ID No 3 or a fragment or a variant thereof.The present invention also embodies isolated, purified, and recombinantpolypeptides comprising a contiguous span of at least 35, 40, 50, 100,150 or 200 amino acids of SEQ ID No 3. The present invention alsoembodies isolated, purified, and recombinant polypeptides comprising acontiguous span of at least 8 amino acids, preferably at least 10 aminoacids, more preferably at least 12, 15, 20, 25, 30, 40, 50, 100, 150 or200 amino acids of SEQ ID No 3, wherein said contiguous span includes atleast 1, 2, 3, 5 or 10 of the following amino acid positions: 1-29,47-70, 103-115, 124, 134, 169-185, and 203-219. Furthermore, the presentinvention embodies isolated, purified, and recombinant polypeptidescomprising a contiguous span of at least 6 amino acids, preferably atleast 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30,40, 50, 100, 150 or 200 amino acids of SEQ ID No 3, wherein saidcontiguous span includes at least 1, 2, 3, 5 or 10 of the followingamino acid positions: 7-11, 18-29, 47, 55-63, 70, 103-104, 111-115, 124,134, 169-173, 181-185, and 203-219.

[0201] Particular regions of the CIDE B polypeptide have interestingfeatures. Two large hydrophilic and antigenic regions respectively beginat the amino acids in position 8 (N) and 79 (E), and respectively end atthe amino acids in position 58 (E) and 146 (L) of the amino acidsequence of CIDE B.

[0202] Four small regions having a good probability to be exposed to theouter environment are the amino acid sequences beginning at positions 31(A), 43 (H), 121 (G), and 138 (V) and respectively ending at positions37 (P), 47 (I), 128 (S) 144 (R) and of the CIDE B protein.

[0203] A further object of the present invention concerns a purified orisolated polypeptide which is encoded by a nucleic acid selected fromthe group consisting of SEQ ID Nos 1 and 2 or fragments or variantsthereof.

[0204] The invention also encompasses a purified or isolated nucleicacid encoding a CIDE B protein having the amino acid sequence of SEQ IDNo 3, or a peptide fragment or variant thereof.

[0205] In a second preferred embodiment, a mutated CIDE B polypeptidecomprises amino acid changes with at least one amino acid deletion,substitution or addition, preferably from 1 to 10, 20 or 30 amino aciddeletions, substitutions or additions. The amino acid substitutions aregenerally non conservative in terms of polarity, charge, hydrophilicityproperties of the substitute amino acid when compared with the nativeamino acid. The amino acid changes occurring in such a mutated CIDE Bpolypeptide may be determinant for the biological activity or for thecapacity of the mutated CIDE B polypeptide to be recognized byantibodies raised against a native CIDE B.

[0206] Such a mutated CIDE B protein may be the target of diagnostictools, such as specific monoclonal or polyclonal antibodies, useful fordetecting the mutated CIDE B protein in a sample.

[0207] The invention also encompasses a CIDE B polypeptide or a fragmentor a variant thereof in which at least one peptide bound has beenmodified as described in the “Definitions” section.

Antibodies that Bind CIDE B Polypeptides of the Invention

[0208] Any CIDE B polypeptide or whole protein may be used to generateantibodies capable of specifically binding to an expressed CIDE Bprotein or fragments thereof as described.

[0209] One antibody composition of the invention is capable ofspecifically binding or specifically bind to the variant of the CIDE Bprotein of SEQ ID No 3. For an antibody composition to specifically bindto CIDE B, it must demonstrate at least a 5%, 10%, 15%, 20%, 25%, 50%,or 100% greater binding affinity for CIDE B protein than for anotherprotein in an ELISA, RIA, or other antibody-based binding assay.

[0210] In a preferred embodiment, the invention concerns antibodycompositions, either polyclonal or monoclonal, capable of selectivelybinding, or selectively bind to an epitope containing a polypeptidecomprising a contiguous span of at least 35, 40, 50, 100, 150 or 200amino acids of SEQ ID No 3; Optionally said epitope contains apolypeptide comprising a contiguous span of at least 8 amino acids,preferably at least 10 amino acids, more preferably at least 12, 15, 20,25, 30, 40, 50, 100, 150 or 200 amino acids of SEQ ID No 3, wherein saidcontiguous span includes at least 1, 2, 3, 5 or 10 of the followingamino acid positions: 1-29, 47-70, 103-115, 124, 134, 169-185, and203-219; Optionally said epitope contains a polypeptide comprising acontiguous span of at least 6 amino acids, preferably at least 8 to 10amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, 100,150 or 200 amino acids of SEQ ID No 3, wherein said contiguous spanincludes at least 1, 2, 3, 5 or 10 of the following amino acidpositions: 7-11, 18-29, 47, 55-63, 70, 103-104, 111-115, 124, 134,169-173, 181-185, and 203-219.

[0211] The invention also concerns a purified or isolated antibodycapable of specifically binding to a mutated CIDE B protein or to afragment or variant thereof comprising an epitope of the mutated CIDE Bprotein. In another preferred embodiment, the present invention concernsan antibody capable of binding to a polypeptide comprising at least 10consecutive amino acids of a CIDE B protein and including at least oneof the amino acids which can be encoded by the trait causing mutations.

[0212] In a preferred embodiment, the invention concerns the use in themanufacture of antibodies of a polypeptide comprising a contiguous spanof at least 35, 40, 50, 100, 150 or 200 amino acids of SEQ ID No 3;Optionally said polypeptide comprises a contiguous span of at least 8amino acids, preferably at least 10 amino acids, more preferably atleast 12, 15, 20, 25, 30, 40, 50, 100, 150 or 200 amino acids of SEQ IDNo 3, wherein said contiguous span includes at least 1, 2, 3, 5 or 10 ofthe following amino acid positions: 1-29, 47-70, 103-115, 124, 134,169-185, and 203-219; Optionally said polypeptide comprises a contiguousspan of at least 6 amino acids, preferably at least 8 to 10 amino acids,more preferably at least 12, 15, 20, 25, 30, 40, 50, 100, 150 or 200amino acids of SEQ ID No 3, wherein said contiguous span includes atleast 1, 2, 3, 5 or 10 of the following amino acid positions: 7-11,18-29, 47, 55-63, 70, 103-104, 111-115, 124, 134, 169-173, 181-185, and203-219.

[0213] The preferred fragments of CIDE B protein used for thepreparation of anti-human CIDE B antibodies comprise, or are comprisedin, the amino acid sequence located between the positions 8 and 58 ofSEQ ID No 3 and between the positions 79 and 146 of SEQ ID No 3. Moreparticularly, the fragments of CIDE B protein used for the preparationof anti-human CIDE B antibodies comprise the amino acid sequence locatedbetween the positions 31 and 37, 43 and 47, 121 and 128, and 138 and 144of the polypeptide sequence of SEQ ID No 3.

[0214] The antibodies of the invention may be labeled by any one of theradioactive, fluorescent or enzymatic labels known in the art.

[0215] The CIDE B polypeptide of SEQ ID No 3 or a fragment thereof canbe used for the preparation of polyclonal or monoclonal antibodies.

[0216] The CIDE B polypeptide expressed from a DNA sequence comprisingat least one of the nucleic acid sequences of SEQ ID Nos 1 and 2 mayalso be used to generate antibodies capable of specifically binding tothe CIDE B polypeptide of SEQ ID No 3 a fragment thereof.

[0217] The antibodies may be prepared from hybridomas according to thetechnique described by Kohler and Milstein in 1975. The polyclonalantibodies may be prepared by immunization of a mammal, especially amouse or a rabbit, with a polypeptide according to the invention that iscombined with an adjuvant of immunity, and then by purifying of thespecific antibodies contained in the serum of the immunized animal on aaffinity chromatography column on which has previously been immobilizedthe polypeptide that has been used as the antigen.

[0218] The present invention also includes, chimeric single chain Fvantibody fragments (Martineau et al., 1998), antibody fragments obtainedthrough phage display libraries (Ridder et al., 1995; Vaughan et al.,1995) and humanized antibodies (Reinmann et al., 1997; Leger et al.,1997).

[0219] Antibody preparations prepared according to either protocol areuseful in quantitative immunoassays which determine concentrations ofantigen-bearing substances in biological samples; they are also usedsemi-quantitatively or qualitatively to identify the presence of antigenin a biological sample. The antibodies may also be used in therapeuticcompositions for killing cells expressing the protein or reducing thelevels of the protein in the body.

[0220] Consequently, the invention is also directed to a method fordetecting specifically the presence of a CIDE B polypeptide according tothe invention in a biological sample, said method comprising thefollowing steps:

[0221] a) bringing into contact the biological sample with a polyclonalor monoclonal antibody that specifically binds a CIDE B polypeptidecomprising an amino acid sequence of SEQ ID No 3, or to a peptidefragment or variant thereof; and

[0222] b) detecting the antigen-antibody complex formed.

[0223] The invention also concerns a diagnostic kit for detecting invitro the presence of a CIDE B polypeptide according to the presentinvention in a biological sample, wherein said kit comprises:

[0224] a) a polyclonal or monoclonal antibody that specifically binds aCIDE B polypeptide comprising an amino acid sequence of SEQ ID No 3, orto a peptide fragment or variant thereof, optionally labeled;

[0225] b) a reagent allowing the detection of the antigen-antibodycomplexes formed, said reagent carrying optionally a label, or beingable to be recognized itself by a labeled reagent, more particularly inthe case when the above-mentioned monoclonal or polyclonal antibody isnot labeled by itself

Biallelic Markers of the CIDE B Gene

[0226] Identification of Biallelic Markers

[0227] There are two preferred methods through which the biallelicmarkers of the present invention can be generated. In a first method,DNA samples from unrelated individuals are pooled together, followingwhich the genomic DNA of interest is amplified and sequenced. Thenucleotide sequences thus obtained are then analyzed to identifysignificant polymorphisms.

[0228] One of the major advantages of this method resides in the factthat the pooling of the DNA samples substantially reduces the number ofDNA amplification reactions and sequencing which must be carried out.Moreover, this method is sufficiently sensitive so that a biallelicmarker obtained therewith usually shows a sufficient degree ofinformativeness for conducting association studies.

[0229] In a second method for generating biallelic markers, the DNAsamples are not pooled and are therefore amplified and sequencedindividually. The resulting nucleotide sequences obtained are then alsoanalyzed to identify significant polymorphisms.

[0230] It will readily be appreciated that when this second method isused, a substantially higher number of DNA amplification reactions mustbe carried out. It will further be appreciated that including suchpotentially less informative biallelic markers in association studies toidentify potential genetic associations with a trait may allow in somecases the direct identification of causal mutations, which may,depending on their penetrance, be rare mutations. This method is usuallypreferred when biallelic markers need to be identified in order toperform association studies within candidate genes.

[0231] In both methods, the genomic DNA samples from which the biallelicmarkers of the present invention are generated are preferably obtainedfrom unrelated individuals corresponding to a heterogeneous populationof known ethnic background, or from familial cases.

[0232] The number of individuals from whom DNA samples are obtained canvary substantially, preferably from about 10 to about 1000, preferablyfrom about 50 to about 200 individuals. It is usually preferred tocollect DNA samples from at least about 100 individuals in order to havesufficient polymorphic diversity in a given population to generate asmany markers as possible and to generate statistically significantresults.

[0233] As for the source of the genomic DNA to be subjected to analysis,any test sample can be foreseen without any particular limitation. Thepreferred source of genomic DNA used in the context of the presentinvention is the peripheral venous blood of each donor.

[0234] The techniques of DNA extraction are well-known to the skilledtechnician. Details of a preferred embodiment are provided in Example 1.

[0235] DNA samples can be pooled or unpooled for the amplification step.DNA amplification techniques are well-known to those skilled in the art.The PCR technology is the preferred amplification technique used in thepresent invention. A typical example of a PCR reaction suitable for thepurposes of the present invention is provided in Example 2.

[0236] The primers used for the amplification are as defined above.Preferred primers of the invention include the nucleotide sequences ofB(12-73), B(12-74), C(12-73), C(12-74), D(12-73-49), D(12-74-38),E(12-73-49), and E(12-74-38). More preferred primers of the inventioninclude the nucleotide sequences of B(12-73), B(12-74), C(12-73), andC(12-74).

[0237] The amplification products generated as described above with theprimers of the invention are then sequenced using methods known andavailable to the skilled technician. Preferably, the amplified DNA issubjected to automated dideoxy terminator sequencing reactions using adye-primer cycle sequencing protocol. Following gel image analysis andDNA sequence extraction, sequence data are automatically processed withadequate software to assess sequence quality.

[0238] The presence of biallelic sites are detected among individual orpooled amplified fragment sequences. Polymorphism search is based on thepresence of superimposed peaks in the electrophoresis pattern. Thesepeaks which present distinct colors correspond to two differentnucleotides at the same position on the sequence. The polymorphism hasto be detected on both strands for validation.

[0239] The biallelic markers of the present invention are disclosed inTable 2 of Example 3. Their location on the CIDE B gene is indicated asfeatures in SEQ ID No 1. The pair of amplification primers are listed inthe sequence listing in features of the SEQ ID No 1 and are described inTable 1 of example 2, these primers allowing the amplification of anucleic acid containing the polymorphic base that defines this biallelicmarker.

[0240] In the present invention, the biallelic markers can be defined bynucleotide sequences corresponding to oligonucleotides of 47 bases inlength comprising at the middle one of the polymorphic base. Moreparticularly, the biallelic markers can be defined by thepolynucleotides P(12-73-49) and P(12-74-38).

[0241] The biallelic markers 12-73-49 and 12-74-38 are located in the 3′regulatory region and form part of the present invention.

[0242] The biallelic markers contained in the human CIDE B gene areuseful tools to perform association studies between the statisticallysignificant occurrence of an allele of said biallelic marker in thegenome of an individual and a specific phenotype, including a phenotypeconsisting of a disorder related to apoptosis such as cancer or AIDS.The biallelic markers of the invention can also be used, for example,for the generation of genetic map, the linkage analysis.

[0243] Genotyping of Biallelic Markers

[0244] Any method known in the art can be used to identify thenucleotide present at a biallelic marker site. Since the biallelicmarker allele to be detected has been identified and specified in thepresent invention, detection will prove simple for one of ordinary skillin the art by employing any of a number of techniques. Many genotypingmethods require the previous amplification of the DNA region carryingthe biallelic marker of interest. While the amplification of target orsignal is often preferred at present, ultrasensitive detection methodswhich do not require amplification are also encompassed by the presentgenotyping methods. Methods well-known to those skilled in the art thatcan be used to detect biallelic polymorphisms include methods such as,conventional dot blot analyzes, single strand conformationalpolymorphism analysis (SSCP) described by Orita et al.(1989), denaturinggradient gel electrophoresis (DGGE), heteroduplex analysis, mismatchcleavage detection, and other conventional techniques as described inSheffield et al.(1991), White et al.(1992), Grompe et al.(1989 and1993). Another method for determining the identity of the nucleotidepresent at a particular polymorphic site employs a specializedexonuclease-resistant nucleotide derivative as described in U.S. Pat.No. 4,656,127.

[0245] Preferred methods involve directly determining the identity ofthe nucleotide present at a biallelic marker site by sequencing assay,enzyme-based mismatch detection assay, or hybridization assay. Thefollowing is a description of some preferred methods. A highly preferredmethod is the microsequencing technique. The term “sequencing” isgenerally used herein to refer to polymerase extension of duplexprimer/template complexes and includes both traditional sequencing andmicrosequencing.

[0246] 1) Sequencing Assays

[0247] The nucleotide present at a polymorphic site can be determined bysequencing methods. In a preferred embodiment, DNA samples are subjectedto PCR amplification before sequencing as described above.

[0248] Preferably, the amplified DNA is subjected to automated dideoxyterminator sequencing reactions using a dye-primer cycle sequencingprotocol. Sequence analysis allows the identification of the basepresent at the biallelic marker site.

[0249] 2) Microsequencing Assays

[0250] In microsequencing methods, the nucleotide at a polymorphic sitein a target DNA is detected by a single nucleotide primer extensionreaction. This method involves appropriate microsequencing primerswhich, hybridize just upstream of the polymorphic base of interest inthe target nucleic acid. A polymerase is used to specifically extend the3′ end of the primer with one single ddNTP (chain terminator)complementary to the nucleotide at the polymorphic site. Next theidentity of the incorporated nucleotide is determined in any suitableway.

[0251] Typically, microsequencing reactions are carried out usingfluorescent ddNTPs and the extended microsequencing primers are analyzedby electrophoresis on ABI 377 sequencing machines to determine theidentity of the incorporated nucleotide as described in EP 412 883.Alternatively capillary electrophoresis can be used in order to processa higher number of assays simultaneously. An example of a typicalmicrosequencing procedure that can be used in the context of the presentinvention is provided in Example 4.

[0252] Different approaches can be used for the labeling and detectionof ddNTPs. A homogeneous phase detection method based on fluorescenceresonance energy transfer has been described by Chen and Kwok (1997) andChen et al.(1997). In this method, amplified genomic DNA fragmentscontaining polymorphic sites are incubated with a 5′-fluorescein-labeledprimer in the presence of allelic dye-labeled dideoxyribonucleosidetriphosphates and a modified Taq polymerase. The dye-labeled primer isextended one base by the dye-terminator specific for the allele presenton the template. At the end of the genotyping reaction, the fluorescenceintensities of the two dyes in the reaction mixture are analyzeddirectly without separation or purification. All these steps can beperformed in the same tube and the fluorescence changes can be monitoredin real time. Alternatively, the extended primer may be analyzed byMALDI-TOF Mass Spectrometry. The base at the polymorphic site isidentified by the mass added onto the microsequencing primer (see Haffand Smirnov, 1997).

[0253] Microsequencing may be achieved by the establishedmicrosequencing method or by developments or derivatives thereof.Alternative methods include several solid-phase microsequencingtechniques. The basic microsequencing protocol is the same as describedpreviously, except that the method is conducted as a heterogeneous phaseassay, in which the primer or the target molecule is immobilized orcaptured onto a solid support. To simplify the primer separation and theterminal nucleotide addition analysis, oligonucleotides are attached tosolid supports or are modified in such ways that permit affinityseparation as well as polymerase extension. The 5′ ends and internalnucleotides of synthetic oligonucleotides can be modified in a number ofdifferent ways to permit different affinity separation approaches, e.g.,biotinylation. If a single affinity group is used on theoligonucleotides, the oligonucleotides can be separated from theincorporated terminator regent. This eliminates the need of physical orsize separation. More than one oligonucleotide can be separated from theterminator reagent and analyzed simultaneously if more than one affinitygroup is used. This permits the analysis of several nucleic acid speciesor more nucleic acid sequence information per extension reaction. Theaffinity group need not be on the priming oligonucleotide but couldalternatively be present on the template. For example, immobilizationcan be carried out via an interaction between biotinylated DNA andstreptavidin-coated microtitration wells or avidin-coated polystyreneparticles. In the same manner, oligonucleotides or templates may beattached to a solid support in a high-density format. In such solidphase microsequencing reactions, incorporated ddNTPs can be radiolabeled(Syvänen, 1994) or linked to fluorescein (Livak and Hainer, 1994). Thedetection of radiolabeled ddNTPs can be achieved throughscintillation-based techniques. The detection of fluorescein-linkedddNTPs can be based on the binding of antifluorescein antibodyconjugated with alkaline phosphatase, followed by incubation with achromogenic substrate (such as p-nitrophenyl phosphate). Other possiblereporter-detection pairs include: ddNTP linked to dinitrophenyl (DNP)and anti-DNP alkaline phosphatase conjugate (Harju et al., 1993) orbiotinylated ddNTP and horseradish peroxidase-conjugated streptavidinwith o-phenylenediamine as a substrate (WO 92/15712). As yet anotheralternative solid-phase microsequencing procedure, Nyren et al.(1993)described a method relying on the detection of DNA polymerase activityby an enzymatic luminometric inorganic pyrophosphate detection assay(ELIDA).

[0254] Pastinen et al.(1997) describe a method for multiplex detectionof single nucleotide polymorphism in which the solid phaseminisequencing principle is applied to an oligonucleotide array format.High-density arrays of DNA probes attached to a solid support (DNAchips) are further described below.

[0255] In one aspect the present invention provides polynucleotides andmethods to genotype one or more biallelic markers of the presentinvention by performing a microsequencing assay. Preferredmicrosequencing primers include the nucleotide sequences D(12-73-49),D(12-74-38), E(12-73-49), and E(12-74-38). It will be appreciated thatthe microsequencing primers listed in Example 4 are merely exemplary andthat, any primer having a 3′ end immediately adjacent to the polymorphicnucleotide may be used. Similarly, it will be appreciated thatmicrosequencing analysis may be performed for any biallelic marker orany combination of biallelic markers of the present invention. Oneaspect of the present invention is a solid support which includes one ormore microsequencing primers listed in Example 4, or fragmentscomprising at least 8, 12, 15, 20, 25, 30, 40, or 50 consecutivenucleotides thereof, to the extent that such lengths are consistent withthe primer described, and having a 3′ terminus immediately upstream ofthe corresponding biallelic marker, for determining the identity of anucleotide at a biallelic marker site.

[0256] 3) Mismatch Detection Assays Based on Polymerases and Ligases

[0257] In one aspect the present invention provides polynucleotides andmethods to determine the allele of one or more biallelic markers of thepresent invention in a biological sample, by mismatch detection assaysbased on polymerases and/or ligases. These assays are based on thespecificity of polymerases and ligases. Polymerization reactions placesparticularly stringent requirements on correct base pairing of the 3′end of the amplification primer and the joining of two oligonucleotideshybridized to a target DNA sequence is quite sensitive to mismatchesclose to the ligation site, especially at the 3′ end. Methods, primersand various parameters to amplify DNA fragments comprising biallelicmarkers of the present invention are further described above in“Amplification Of DNA Fragments Comprising Biallelic Markers”.

[0258] Allele Specific Amplification Primers

[0259] Discrimination between the two alleles of a biallelic marker canalso be achieved by allele specific amplification, a selective strategy,whereby one of the alleles is amplified without amplification of theother allele. For allele specific amplification, at least one member ofthe pair of primers is sufficiently complementary with a region of aCIDE B gene comprising the polymorphic base of a biallelic marker of thepresent invention to hybridize therewith and to initiate theamplification. Such primers are able to discriminate between the twoalleles of a biallelic marker.

[0260] This is accomplished by placing the polymorphic base at the 3′end of one of the amplification primers. Because the extension formsfrom the 3′ end of the primer, a mismatch at or near this position hasan inhibitory effect on amplification. Therefore, under appropriateamplification conditions, these primers only direct amplification ontheir complementary allele. Determining the precise location of themismatch and the corresponding assay conditions are well within theordinary skill in the art.

[0261] Ligation/Amplification Based Methods

[0262] The “Oligonucleotide Ligation Assay” (OLA) uses twooligonucleotides which are designed to be capable of hybridizing toabutting sequences of a single strand of a target molecules. One of theoligonucleotides is biotinylated, and the other is detectably labeled.If the precise complementary sequence is found in a target molecule, theoligonucleotides will hybridize such that their termini abut, and createa ligation substrate that can be captured and detected. OLA is capableof detecting single nucleotide polymorphisms and may be advantageouslycombined with PCR as described by Nickerson et al.(1990). In thismethod, PCR is used to achieve the exponential amplification of targetDNA, which is then detected using OLA.

[0263] Other amplification methods which are particularly suited for thedetection of single nucleotide polymorphism include LCR (ligase chainreaction), Gap LCR (GLCR) which are described above in “DNAAmplification”. LCR uses two pairs of probes to exponentially amplify aspecific target. The sequences of each pair of oligonucleotides, isselected to permit the pair to hybridize to abutting sequences of thesame strand of the target. Such hybridization forms a substrate for atemplate-dependant ligase. In accordance with the present invention, LCRcan be performed with oligonucleotides having the proximal and distalsequences of the same strand of a biallelic marker site. In oneembodiment, either oligonucleotide will be designed to include thebiallelic marker site. In such an embodiment, the reaction conditionsare selected such that the oligonucleotides can be ligated together onlyif the target molecule either contains or lacks the specific nucleotidethat is complementary to the biallelic marker on the oligonucleotide. Inan alternative embodiment, the oligonucleotides will not include thebiallelic marker, such that when they hybridize to the target molecule,a “gap” is created as described in WO 90/01069. This gap is then“filled” with complementary dNTPs (as mediated by DNA polymerase), or byan additional pair of oligonucleotides. Thus at the end of each cycle,each single strand has a complement capable of serving as a targetduring the next cycle and exponential allele-specific amplification ofthe desired sequence is obtained.

[0264] Ligase/Polymerase-mediated Genetic Bit Analysis™ is anothermethod for determining the identity of a nucleotide at a preselectedsite in a nucleic acid molecule (WO 95/21271). This method involves theincorporation of a nucleoside triphosphate that is complementary to thenucleotide present at the preselected site onto the terminus of a primermolecule, and their subsequent ligation to a second oligonucleotide. Thereaction is monitored by detecting a specific label attached to thereaction's solid phase or by detection in solution.

[0265] 4) Hybridization Assay Methods

[0266] A preferred method of determining the identity of the nucleotidepresent at a biallelic marker site involves nucleic acid hybridization.The hybridization probes, which can be conveniently used in suchreactions, preferably include the probes defined herein. Anyhybridization assay may be used including Southern hybridization,Northern hybridization, dot blot hybridization and solid-phasehybridization (see Sambrook et al., 1989).

[0267] Hybridization refers to the formation of a duplex structure bytwo single stranded nucleic acids due to complementary base pairing.Hybridization can occur between exactly complementary nucleic acidstrands or between nucleic acid strands that contain minor regions ofmismatch. Specific probes can be designed that hybridize to one form ofa biallelic marker and not to the other and therefore are able todiscriminate between different allelic forms. Allele-specific probes areoften used in pairs, one member of a pair showing perfect match to atarget sequence containing the original allele and the other showing aperfect match to the target sequence containing the alternative allele.Hybridization conditions should be sufficiently stringent that there isa significant difference in hybridization intensity between alleles, andpreferably an essentially binary response, whereby a probe hybridizes toonly one of the alleles. Stringent, sequence specific hybridizationconditions, under which a probe will hybridize only to the exactlycomplementary target sequence are well known in the art (Sambrook etal., 1989). Stringent conditions are sequence dependent and will bedifferent in different circumstances. Generally, stringent conditionsare selected to be about 5° C. lower than the thermal melting point (Tm)for the specific sequence at a defined ionic strength and pH. Althoughsuch hybridization can be performed in solution, it is preferred toemploy a solid-phase hybridization assay. The target DNA comprising abiallelic marker of the present invention may be amplified prior to thehybridization reaction. The presence of a specific allele in the sampleis determined by detecting the presence or the absence of stable hybridduplexes formed between the probe and the target DNA. The detection ofhybrid duplexes can be carried out by a number of methods. Variousdetection assay formats are well known which utilize detectable labelsbound to either the target or the probe to enable detection of thehybrid duplexes. Typically, hybridization duplexes are separated fromunhybridized nucleic acids and the labels bound to the duplexes are thendetected. Those skilled in the art will recognize that wash steps may beemployed to wash away excess target DNA or probe as well as unboundconjugate. Further, standard heterogeneous assay formats are suitablefor detecting the hybrids using the labels present on the primers andprobes.

[0268] Two recently developed assays allow hybridization-based allelediscrimination with no need for separations or washes (see Landegren U.et al., 1998). The TaqMan assay takes advantage of the 5′ nucleaseactivity of Taq DNA polymerase to digest a DNA probe annealedspecifically to the accumulating amplification product. TaqMan probesare labeled with a donor-acceptor dye pair that interacts viafluorescence energy transfer. Cleavage of the TaqMan probe by theadvancing polymerase during amplification dissociates the donor dye fromthe quenching acceptor dye, greatly increasing the donor fluorescence.All reagents necessary to detect two allelic variants can be assembledat the beginning of the reaction and the results are monitored in realtime (see Livak et al., 1995). In an alternative homogeneoushybridization based procedure, molecular beacons are used for allelediscriminations. Molecular beacons are hairpin-shaped oligonucleotideprobes that report the presence of specific nucleic acids in homogeneoussolutions. When they bind to their targets they undergo a conformationalreorganization that restores the fluorescence of an internally quenchedfluorophore (Tyagi et al., 1998).

[0269] The polynucleotides provided herein can be used to produce probeswhich can be used in hybridization assays for the detection of biallelicmarker alleles in biological samples. These probes are characterized inthat they preferably comprise between 8 and 50 nucleotides, and in thatthey are sufficiently complementary to a sequence comprising a biallelicmarker of the present invention to hybridize thereto and preferablysufficiently specific to be able to discriminate the targeted sequencefor only one nucleotide variation. A particularly preferred probe is 25nucleotides in length. Preferably the biallelic marker is within 4nucleotides of the center of the polynucleotide probe. In particularlypreferred probes, the biallelic marker is at the center of saidpolynucleotide. Preferred probes comprise a nucleotide sequence selectedfrom the group consisting of amplicons listed in Table 1 and thesequences complementary thereto, or a fragment thereof, said fragmentcomprising at least about 8 consecutive nucleotides, preferably 10, 15,20, more preferably 25, 30, 40, 47, or 50 consecutive nucleotides andcontaining a polymorphic base. Preferred probes comprise anucleotidesequence selected from the group consisting of P(12-73-49) andP(12-74-38) and the sequences complementary thereto. In preferredembodiments the polymorphic base(s) are within 5, 4, 3, 2, 1,nucleotides of the center of the said polynucleotide, more preferably atthe center of said polynucleotide.

[0270] Preferably the probes of the present invention are labeled orimmobilized on a solid support. Labels and solid supports are furtherdescribed in “Oligonucleotide Probes and Primers”. The probes can benon-extendable as described in “Oligonucleotide Probes and Primers”.

[0271] By assaying the hybridization to an allele specific probe, onecan detect the presence or absence of a biallelic marker allele in agiven sample. High-Throughput parallel hybridization in array format isspecifically encompassed within “hybridization assays” and are describedbelow.

[0272] 5) Hybridization to Addressable Arrays of Oligonucleotides

[0273] Hybridization assays based on oligonucleotide arrays rely on thedifferences in hybridization stability of short oligonucleotides toperfectly matched and mismatched target sequence variants. Efficientaccess to polymorphism information is obtained through a basic structurecomprising high-density arrays of oligonucleotide probes attached to asolid support (e.g., the chip) at selected positions. Each DNA chip cancontain thousands to millions of individual synthetic DNA probesarranged in a grid-like pattern and miniaturized to the size of a dime.

[0274] The chip technology has already been applied with success innumerous cases. For example, the screening of mutations has beenundertaken in the BRCA1 gene, in S. cerevisiae mutant strains, and inthe protease gene of HIV-1 virus (Hacia et al., 1996; Shoemaker et al.,1996; Kozal et al., 1996). Chips of various formats for use in detectingbiallelic polymorphisms can be produced on a customized basis byAffymetrix (GeneChip™), Hyseq (HyChip and HyGnostics), and ProtogeneLaboratories.

[0275] In general, these methods employ arrays of oligonucleotide probesthat are complementary to target nucleic acid sequence segments from anindividual which, target sequences include a polymorphic marker. EP785280 describes a tiling strategy for the detection of singlenucleotide polymorphisms. Briefly, arrays may generally be “tiled” for alarge number of specific polymorphisms. By “tiling” is generally meantthe synthesis of a defined set of oligonucleotide probes which is madeup of a sequence complementary to the target sequence of interest, aswell as preselected variations of that sequence, e.g., substitution ofone or more given positions with one or more members of the basis set ofnucleotides. Tiling strategies are further described in PCT applicationNo. WO 95/11995. In a particular aspect, arrays are tiled for a numberof specific, identified biallelic marker sequences. In particular, thearray is tiled to include a number of detection blocks, each detectionblock being specific for a specific biallelic marker or a set ofbiallelic markers. For example, a detection block may be tiled toinclude a number of probes, which span the sequence segment thatincludes a specific polymorphism. To ensure probes that arecomplementary to each allele, the probes are synthesized in pairsdiffering at the biallelic marker. In addition to the probes differingat the polymorphic base, monosubstituted probes are also generally tiledwithin the detection block. These monosubstituted probes have bases atand up to a certain number of bases in either direction from thepolymorphism, substituted with the remaining nucleotides (selected fromA, T, G, C and U). Typically the probes in a tiled detection block willinclude substitutions of the sequence positions up to and includingthose that are 5 bases away from the biallelic marker. Themonosubstituted probes provide internal controls for the tiled array, todistinguish actual hybridization from artefactual cross-hybridization.Upon completion of hybridization with the target sequence and washing ofthe array, the array is scanned to determine the position on the arrayto which the target sequence hybridizes. The hybridization data from thescanned array is then analyzed to identify which allele or alleles ofthe biallelic marker are present in the sample. Hybridization andscanning may be carried out as described in PCT application No. WO92/10092 and WO 95/11995 and U.S. Pat. No. 5,424,186.

[0276] Thus, in some embodiments, the chips may comprise an array ofnucleic acid sequences of fragments of about 15 nucleotides in length.In further embodiments, the chip may comprise an array including atleast one of the sequences selected from the group consisting ofamplicons listed in table 1 and the sequences complementary thereto, ora fragment thereof, said fragment comprising at least about 8consecutive nucleotides, preferably 10, 15, 20, more preferably 25, 30,40, 47, or 50 consecutive nucleotides and containing a polymorphic base.In preferred embodiments the polymorphic base is within 5, 4, 3, 2, 1,nucleotides of the center of the said polynucleotide, more preferably atthe center of said polynucleotide. In some embodiments, the chip maycomprise an array of at least 2, 3, 4, 5,6, 7, 8 or more of thesepolynucleotides of the invention. Solid supports and polynucleotides ofthe present invention attached to solid supports are further describedin “Oligonucleotide Probes And Primers”.

[0277] 6) Integrated Systems

[0278] Another technique, which may be used to analyze polymorphisms,includes multicomponent integrated systems, which miniaturize andcompartmentalize processes such as PCR and capillary electrophoresisreactions in a single functional device. An example of such technique isdisclosed in U.S. Pat. No. 5,589,136, which describes the integration ofPCR amplification and capillary electrophoresis in chips.

[0279] Integrated systems can be envisaged mainly when microfluidicsystems are used. These systems comprise a pattern of microchannelsdesigned onto a glass, silicon, quartz, or plastic wafer included on amicrochip. The movements of the samples are controlled by electric,electroosmotic or hydrostatic forces applied across different areas ofthe microchip to create functional microscopic valves and pumps with nomoving parts.

[0280] For genotyping biallelic markers, the microfluidic system mayintegrate nucleic acid amplification, microsequencing, capillaryelectrophoresis and a detection method such as laser-inducedfluorescence detection.

[0281] Expression of a Regulatory or Coding Polynucleotide of CIDE B.

[0282] Any of the regulatory polynucleotides or the codingpolynucleotides of the invention may be inserted into recombinantvectors for expression in a recombinant host cell or a recombinant hostorganism.

[0283] Thus, the present invention also encompasses a family ofrecombinant vectors that contains either a regulatory polynucleotideselected from the group consisting of any one of the regulatorypolynucleotides derived from the CIDE B genomic sequence, or a codingpolynucleotide from the CIDE B genomic sequence. Consequently, thepresent invention further deals with a recombinant vector comprisingeither a regulatory polynucleotide contained in the nucleic acid of SEQID No 1, or a polynucleotide comprising the CIDE B coding sequence, orboth.

[0284] In a first preferred embodiment, a recombinant vector of theinvention is used as an expression vector; (a) the CIDE B regulatorysequence comprised therein drives the expression of a codingpolynucleotide operably linked thereto; (b) the CIDE B coding sequenceis operably linked to regulation sequences allowing its expression in asuitable cell host and/or host organism.

[0285] In a second preferred embodiment, a recombinant vector of theinvention is used to amplify the inserted polynucleotide derived from aCIDE B genomic sequence selected from the group consisting of thenucleic acids of SEQ ID No 1 or a CIDE B cDNA in a suitable cell host,this polynucleotide being amplified at every time that the recombinantvector replicates.

[0286] More particularly, the present invention relates to expressionvectors which include nucleic acids encoding a CIDE B protein,preferably the CIDE B protein of the amino acid sequence of SEQ ID No 3or variants or fragments thereof, under the control of a regulatorysequence selected among the CIDE B regulatory polynucleotides, oralternatively under the control of an exogenous regulatory sequence.

[0287] A recombinant expression vector comprising a nucleic acidselected from the group consisting of the 5′ regulatory region, orbiologically active fragments or variants thereof, is also part of thepresent invention.

[0288] Generally, a recombinant vector of the invention may comprise anyof the polynucleotides described herein, including regulatory sequences,and coding sequences, as well as any CIDE B primer or probe as definedabove. More particularly, the recombinant vectors of the presentinvention can comprise any of the polynucleotides described in the “CIDEB cDNA Sequences” section, the “Coding Regions of CIDE B” section,“Genomic sequence of CIDE B” section and the “Oligonucleotide Probes AndPrimers” section.

[0289] Some of the elements which can be found in the vectors of thepresent invention are described in further detail in the followingsections.

[0290] Vectors

[0291] A recombinant vector according to the invention comprises, but isnot limited to, a YAC (Yeast Artificial Chromosome), a BAC (BacterialArtificial Chromosome), a phage, a phagemid, a cosmid, a plasmid or evena linear DNA molecule which may consist of a chromosomal,non-chromosomal and synthetic DNA. Such a recombinant vector cancomprise a transcriptional unit comprising an assembly of:

[0292] (1) a genetic element or elements having a regulatory role ingene expression, for example promoters or enhancers. Enhancers arecis-acting elements of DNA, usually from about 10 to 300 bp in lengththat act on the promoter to increase the transcription.

[0293] (2) a structural or coding sequence which is transcribed intomRNA and eventually translated into a polypeptide, and

[0294] (3) appropriate transcription initiation and terminationsequences. Structural units intended for use in yeast or eukaryoticexpression systems preferably include a leader sequence enablingextracellular secretion of translated protein by a host cell.Alternatively, where a recombinant protein is expressed without a leaderor transport sequence, it may include an N-terminal residue. Thisresidue may or may not be subsequently cleaved from the expressedrecombinant protein to provide a final product.

[0295] Generally, recombinant expression vectors will include origins ofreplication, selectable markers permitting transformation of the hostcell, and a promoter derived from a highly expressed gene to directtranscription of a downstream structural sequence. The heterologousstructural sequence is assembled in appropriate phase with translationinitiation and termination sequences, and preferably a leader sequencecapable of directing secretion of the translated protein into theperiplasmic space or the extracellular medium.

[0296] The selectable marker genes for selection of transformed hostcells are preferably dihydrofolate reductase or neomycin resistance foreukaryotic cell culture, TRP1 for S. cerevisiae or tetracycline,rifampicin or ampicillin resistance in E. coli, or levan saccharase formycobacteria.

[0297] As a representative but non-limiting example, useful expressionvectors for bacterial use can comprise a selectable marker and abacterial origin of replication derived from commercially availableplasmids comprising genetic elements of pBR322 (ATCC 37017). Suchcommercial vectors include, for example, pKK223-3 (Pharmacia, Uppsala,Sweden), and GEM1 (Promega Biotec, Madison, Wis., USA).

[0298] Large numbers of suitable vectors and promoters are known tothose of skill in the art, and commercially available, such as bacterialvectors: pQE70, pQE60, pQE9 (Qiagen), pbs, pD10, phagescript, psiX174,pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene);ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); or eukaryoticvectors pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV,pMSG, pSVL (Pharmacia); baculovirus transfer vector pVL1392/1393(Pharmingen); pQE-30 (QIAexpress).

[0299] A suitable vector for the expression of the CIDE B polypeptide ofSEQ ID No 3 or fragments or variants thereof is a baculovirus vectorthat can be propagated in insect cells and in insect cell lines. Aspecific suitable host vector system is the pVL1392/1393 baculovirustransfer vector (Pharmingen) that is used to transfect the SF9 cell line(ATCC N^(o)CRL 1711) which is derived from Spodoptera frugiperda.

[0300] Other suitable vectors for the expression of the CIDE Bpolypeptide of SEQ ID No 3 or fragments or variants thereof in abaculovirus expression system include those described by Chai et al.(1993), Vlasak et al. (1983) and Lenhard et al. (1996).

[0301] Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′-flankingnon-transcribed sequences. DNA sequences derived from the SV40 viralgenome, for example SV40 origin, early promoter, enhancer, splice andpolyadenylation sites may be used to provide the requirednon-transcribed genetic elements.

[0302] Promoters

[0303] The suitable promoter regions used in the expression vectorsaccording to the present invention are chosen taking into account thecell host in which the heterologous gene has to be expressed.

[0304] A suitable promoter may be heterologous with respect to thenucleic acid for which it controls the expression or alternatively canbe endogenous to the native polynucleotide containing the codingsequence to be expressed. Additionally, the promoter is generallyheterologous with respect to the recombinant vector sequences withinwhich the construct promoter/coding sequence has been inserted.

[0305] Preferred bacterial promoters are the LacI, LacZ, the T3 or T7bacteriophage RNA polymerase promoters, the polyhedrin promoter, or thep10 protein promoter from baculovirus (Kit Novagen) (Smith et al., 1983;O'Reilly et al., 1992), the lambda PR promoter or also the trc promoter.

[0306] Promoter regions can be selected from any desired gene using, forexample, CAT (chloramphenicol transferase) vectors and more preferablypKK232-8 and pCM7 vectors. Particularly preferred bacterial promotersinclude lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryoticpromoters include CMV immediate early, HSV thymidine kinase, early andlate SV40, LTRs from retrovirus, and mouse metallothionein-L. Selectionof a convenient vector and promoter is well within the level of ordinaryskill in the art.

[0307] The choice of a promoter is well within the ability of a personskilled in the field of genetic egineering. For example, one may referto the book of Sambrook et al. (1989) or also to the proceduresdescribed by Fuller et al. (1996).

[0308] The vector containing the appropriate DNA sequence as describedabove, more preferably CIDE B gene regulatory polynucleotide, apolynucleotide encoding the CIDE B polypeptide of SEQ ID No 3 or both ofthem, can be utilized to transform an appropriate host to allow theexpression of the desired polypeptide or polynucleotide.

[0309] Other Types of Vectors

[0310] The in vivo expression of a CIDE B polypeptide of SEQ ID No No 3or fragments or variants thereof may be useful in order to correct agenetic defect related to the expression of the native gene in a hostorganism or to the production of a biologically inactive CIDE B protein.

[0311] Consequently, the present invention also deals with recombinantexpression vectors mainly designed for the in vivo production of theCIDE B polypeptide of SEQ ID No No 3 or fragments or variants thereof bythe introduction of the appropriate genetic material in the organism ofthe patient to be treated. This genetic material may be introduced invitro in a cell that has been previously extracted from the organism,the modified cell being subsequently reintroduced in the said organism,directly in vivo into the appropriate tissue.

[0312] By <<vector>> according to this specific embodiment of theinvention is intended either a circular or a linear DNA molecule.

[0313] One specific embodiment for a method for delivering a protein orpeptide to the interior of a cell of a vertebrate in vivo comprises thestep of introducing a preparation comprising a physiologicallyacceptable carrier and a naked polynucleotide operatively coding for thepolypeptide of interest into the interstitial space of a tissuecomprising the cell, whereby the naked polynucleotide is taken up intothe interior of the cell and has a physiological effect.

[0314] In a specific embodiment, the invention provides a compositionfor the in vivo production of the CIDE B protein or polypeptidedescribed herein. It comprises a naked polynucleotide operatively codingfor this polypeptide, in solution in a physiologically acceptablecarrier, and suitable for introduction into a tissue to cause cells ofthe tissue to express the said protein or polypeptide.

[0315] Compositions comprising a polynucleotide are described in PCTapplication N^(o) WO 90/11092 (Vical Inc.) and also in PCT applicationN^(o) WO 95/11307 (Institut Pasteur, INSERM, Universitéd'Ottawa) as wellas in the articles of Tacson et al. (1996) and of Huygen et al. (1996).

[0316] The amount of vector to be injected to the desired host organismvaries according to the site of injection. As an indicative dose, itwill be injected between 0,1 and 100 μg of the vector in an animal body,preferably a mammal body, for example a mouse body.

[0317] In another embodiment of the vector according to the invention,it may be introduced in vitro in a host cell, preferably in a host cellpreviously harvested from the animal to be treated and more preferably asomatic cell such as a muscle cell. In a subsequent step, the cell thathas been transformed with the vector coding for the desired CIDE Bpolypeptide or the desired fragment thereof is reintroduced into theanimal body in order to deliver the recombinant protein within the bodyeither locally or systemically.

[0318] In one specific embodiment, the vector is derived from anadenovirus. Preferred adenovirus vectors according to the invention arethose described by Feldman and Steg (1996) or Ohno et al. (1994).Another preferred recombinant adenovirus according to this specificembodiment of the present invention is the human adenovirus type 2 or 5(Ad 2 or Ad 5) or an adenovirus of animal origin ( French patentapplication N^(o) FR-93.05954).

[0319] Retrovirus vectors and adeno-associated virus vectors aregenerally understood to be the recombinant gene delivery systems ofchoice for the transfer of exogenous polynucleotides in vivo,particularly to mammals, including humans. These vectors provideefficient delivery of genes into cells, and the transferred nucleicacids are stably integrated into the chromosomal DNA of the host

[0320] Particularly preferred retroviruses for the preparation orconstruction of retroviral in vitro or in vitro gene delivery vehiclesof the present invention include retroviruses selected from the groupconsisting of Mink-Cell Focus Inducing Virus, Murine Sarcoma Virus,Reticuloendotheliosis virus and Rous Sarcoma virus. Particularlypreferred Murine Leukemia Viruses include the 4070A and the 1504Aviruses, Abelson (ATCC No VR-999), Friend (ATCC No VR-245), Gross (ATCCNo VR-590), Rauscher (ATCC No VR-998) and Moloney Murine Leukemia Virus(ATCC No VR-190; PCT Application No WO 94/24298). Particularly preferredRous Sarcoma Viruses include Bryan high titer (ATCC Nos VR-334, VR-657,VR-726, VR-659 and VR-728). Other preferred retroviral vectors are thosedescribed in Roth et al. (1996), PCT Application No WO 93/25234, PCTApplication No WO 94/06920, Roux et al., 1989, Julan et al., 1992 andNeda et al., 1991.

[0321] Yet another viral vector system that is contemplated by theinvention consists in the adeno-associated virus (AAV). Theadeno-associated virus is a naturally occurring defective virus thatrequires another virus, such as an adenovirus or a herpes virus, as ahelper virus for efficient replication and a productive life cycle(Muzyczka et al., 1992). It is also one of the few viruses that mayintegrate its DNA into non-dividing cells, and exhibits a high frequencyof stable integration (Flotte et al., 1992; Samulski et al., 1989;McLaughlin et al., 1989). One advantageous feature of AAV derives fromits reduced efficacy for transducing primary cells relative totransformed cells.

[0322] Other compositions containing a vector of the inventionadvantageously comprise an oligonucleotide fragment of the nucleicsequence SEQ ID No 2, preferably a fragment including the start codon ofthe CIDE B gene, as an antisense tool that inhibits the expression ofthe corresponding CIDE B gene. Preferred methods using antisensepolynucleotide according to the present invention are the proceduresdescribed by Sczakiel et al. (1995) or those described in PCTApplication No WO 95/24223.

[0323] Preferably, the antisense tools are chosen among thepolynucleotides (15-200 bp long) that are complementary to the 5′end ofthe CIDE B mRNA. In another embodiment, a combination of differentantisense polynucleotides complementary to different parts of thedesired targeted gene are used.

[0324] Preferred antisense polynucleotides according to the presentinvention are complementary to a sequence of the mRNAs of CIDE B thatcontains the translation initiation codon ATG.

[0325] Host Cells

[0326] Another object of the invention consists in host cell that havebeen transformed or transfected with one of the polynucleotidesdescribed therein, and more precisely a polynucleotide either comprisinga CIDE B regulatory polynucleotide or the coding sequence of the CIDE Bpolypeptide having the amino acid sequence of SEQ ID No 3 or fragmentsor variants thereof. Are included host cells that are transformed(prokaryotic cells) or that are transfected (eukaryotic cells) with arecombinant vector such as one of those described above.

[0327] A recombinant host cell of the invention comprises any one of thepolynucleotides or the recombinant vectors described therein. Moreparticularly, the cell hosts of the present invention can comprise anyof the polynucleotides described in “CIDEB cDNA Sequences” section, the“Coding Regions Of CIDE B” section, “Genomic sequence of CIDE B”section, the “Oligonucleotide Probes And Primers” section and the“Vectors for the expression of a regulatory or coding polynucleotide ofCIDE B” section.

[0328] Preferred host cells used as recipients for the expressionvectors of the invention are the following:

[0329] a) Prokaryotic host cells: Escherichia coli strains (I.E. DH5-αstrain) or Bacillus subtilis.

[0330] b) Eukaryotic host cells : HeLa cells (ATCC N^(o)CCL2;N^(o)CCL2.1; N^(o)CCL2.2), Cv 1 cells (ATCC N^(o)CCL70), COS cells (ATCCN^(o)CRL1650; N^(o)CRL1651), Sf-9 cells (ATCC N^(o)CRL1711).

[0331] The constructs in the host cells can be used in a conventionalmanner to produce the gene product encoded by the recombinant sequence.

[0332] Following transformation of a suitable host and growth of thehost to an appropriate cell density, the selected promoter is induced byappropriate means, such as temperature shift or chemical induction, andcells are cultivated for an additional period.

[0333] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

[0334] Microbial cells employed in the expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents. Suchmethods are well known by the skill artisan.

[0335] Transgenic Animals

[0336] The terms “transgenic animals” or “host animals” are used hereinto designate animals that have their genome genetically and artificiallymanipulated so as to include one of the nucleic acids according to theinvention. Preferred animals are non-human mammals and include thosebelonging to a genus selected from Mus (e.g. mice), Rattus (e.g. rats)and Oryctogalus (e.g. rabbits) which have their genome artificially andgenetically altered by the insertion of a nucleic acid according to theinvention.

[0337] The transgenic animals of the invention all include within aplurality of their cells a cloned recombinant or synthetic DNA sequence,more specifically one of the purified or isolated nucleic acidscomprising a CIDE B coding sequence, a CIDE B regulatory polynucleotideor a DNA sequence encoding an antisense polynucleotide such as describedin the present specification.

[0338] More particularly, transgenic animals according to the inventioncontain in their somatic cells and/or in their germ line cells any ofthe polynucleotides described in “CIDE B cDNA Sequences” section, the“Coding Regions Of CIDE B” section, “Genomic sequence of CIDE B”section, the “Oligonucleotide Probes And Primers” section and the“Vectors for the expression of a regulatory or coding polynucleotide ofCIDE B” section.

[0339] The replacement of the native genomic CIDE B sequence by adefective copy of said sequence may be preformed by techniques of genetargeting. Such techniques are notably described by Burright et al.(1997), Bates et al. (1997), Mangiarini et al. (1997), Davies et al.(1997).

[0340] Second preferred transgenic animals of the invention have themurine CIDE B gene replaced either by a defective copy of the murineCIDE B gene or by an interrupted copy of the human CIDE B gene. A“defective copy” of a murine or a human CIDE B gene, is intended todesignate a modified copy of these genes that is not or poorlytranscribed in the resulting recombinant host animal or a modified copyof these genes leading to the absence of synthesis of the correspondingtranslation product or alternatively leading to a modified and/ortruncated translation product lacking the biological activity of thewild type CIDE B protein. The altered translation product thus containsamino acid modifications, deletions and substitutions. Modifications anddeletions may render the naturally occurring gene nonfunctional, thusleading to a “knockout animal”. These transgenic animals are criticalfor the creation of animal models of human diseases, and for eventualtreatment of disorders related to apoptosis such as cancer or AIDS.Examples of such knockout mice are described in the PCT Applications NosWO 97/34641, WO 96/12792 and WO 98/02354.

[0341] The endogenous murine CIDE B gene can be interrupted by theinsertion, between two contiguous nucleotide of said gene, of a part ofall of a marker gene placed under the control of the appropriatepromoter, for example the endogenous promoter of the endogenous murineCIDE B gene. The marker gene may be the neomycin resistance gene (neo)that may be operably linked to the phosphoglycerate kinase-1 (PGK-1)promoter, as described in the PCT Application No WO 98/02534.

[0342] Thus, the invention is also directed to a transgenic animalcontain in their somatic cells and/or in their germ line cells apolynucleotide selected from the following group of polynucleotides:

[0343] a) a defective copy of the human CIDE B gene;

[0344] b) a defective copy of the endogenous CIDE B gene, wherein theexpression “endogenous CIDE B gene” designates a CIDE B gene that isnaturally present within the genome of the animal host to be geneticallymodified.

[0345] The invention also concerns a method for obtaining transgenicanimals, wherein said methods comprise the steps of:

[0346] a) replacing the endogenous copy of the animal CIDE B gene by anucleic acid selected from the group consisting of a defective copy ofthe human CIDE B gene and a defective copy of the endogenous CIDE B genein animal cells, preferably embryonic stem cells (ES);

[0347] b) introducing the recombinant animal cells obtained at step a)in embryos, notably blastocysts of the animal;

[0348] c) selecting the resulting transgenic animals, for example bydetecting the defective copy of a CIDE B gene with one or severalprimers or probes according to the invention.

[0349] Optionally, the transgenic animals may be bred together in orderto obtain homozygous transgenic animals for the defective copy of theCIDE B gene introduced.

[0350] The transgenic animals of the invention thus contain specificsequences of exogenous genetic material such as the nucleotide sequencesdescribed above in detail.

[0351] In a first preferred embodiment, these transgenic animals may begood experimental models in order to study the diverse pathologiesrelated to disorders associated to apoptosis, in particular concerningthe transgenic animals within the genome of which has been inserted oneor several copies of a polynucleotide encoding a native CIDE B protein,or alternatively a mutant CIDE B protein.

[0352] In a second preferred embodiment, these transgenic animals mayexpress a desired polypeptide of interest under the control of theregulatory polynucleotides of the CIDE B gene, leading to good yields inthe synthesis of this protein of interest, and eventually a tissuespecific expression of this protein of interest.

[0353] Since it is possible to produce transgenic animals of theinvention using a variety of different sequences, a general descriptionwill be given of the production of transgenic animals by referringgenerally to exogenous genetic material. This general description can beadapted by those skilled in the art in order to incorporate the DNAsequences into animals. For more details regarding the production oftransgenic animals, and specifically transgenic mice, it may be referredto Sandou et al. (1994) and also to U.S. Pat. No. 4,873,191, issued Oct.10, 1989, U.S. Pat. No. 5,968,766, issued Dec. 16, 1997 and U.S. Pat.No. 5,387,742, issued Feb. 28, 1995.

[0354] Transgenic animals of the present invention are produced by theapplication of procedures which result in an animal with a genome thatincorporates exogenous genetic material which is integrated into thegenome. The procedure involves obtaining the genetic material or aportion thereof, which encodes either a CIDE B coding sequence, a CIDE Bregulatory polynucleotide or a DNA sequence encoding an antisensepolynucleotide such as described in the present specification.

[0355] A recombinant polynucleotide of the invention is inserted into anembryonic or ES stem cell line. The insertion is made usingelectroporation. The cells subjected to electroporation are screened(e.g. Southern blot analysis) to find positive cells which haveintegrated the exogenous recombinant polynucleotide into their genome.An illustrative positive-negative selection procedure that may be usedaccording to the invention is described by Mansour et al. (1988). Then,the positive cells are isolated, cloned and injected into 3.5 days oldblastocysts from mice. The blastocysts are then inserted into a femalehost animal and allowed to grow to term. The offsprings of the femalehost are tested to determine which animals are transgenic e.g. includethe inserted exogenous DNA sequence and which are wild-type.

[0356] Thus, the present invention also concerns a transgenic animalcontaining a nucleic acid, a recombinant expression vector or arecombinant host cell according to the invention.

[0357] Methods for Screening Substances Interacting with a CIDE BPolypeptide

[0358] For the purpose of the present invention, a ligand means amolecule, such as a protein, a peptide, an antibody or any syntheticchemical compound capable of binding to the CIDE B protein or one of itsfragments or variants or to modulate the expression of thepolynucleotide coding for CIDE B or a fragment or variant thereof.

[0359] In the ligand screening method according to the presentinvention, a biological sample or a defined molecule to be tested as aputative ligand of the CIDE B protein is brought into contact with thepurified CIDE B protein, for example the purified recombinant CIDE Bprotein produced by a recombinant cell host as described hereinbefore,in order to form a complex between the CIDE B protein and the putativeligand molecule to be tested.

[0360] Another object of the present invention consists of methods andkits for the screening of candidate substances that interact with a CIDEB polypeptide.

[0361] The present invention pertains to methods for screeningsubstances of interest that interact with a CIDE B protein or onefragment or variant thereof. By their capacity to bind covalently ornon-covalently to a CIDE B protein or to a fragment or variant thereof,these substances or molecules may be advantageously used both in vitroand in vivo.

[0362] In vitro, said interacting molecules may be used as detectionmeans in order to identify the presence of a CIDE B protein in a sample,preferably a biological sample.

[0363] A method for the screening of a candidate substance interactingwith a CIDE B polypeptide of the present invention comprises thefollowing steps:

[0364] a) providing a polypeptide consisting of a CIDE B protein or afragment or a variant thereof;

[0365] b) obtaining a candidate substance;

[0366] c) bringing into contact said polypeptide with said candidatesubstance;

[0367] d) detecting the complexes formed between said polypeptide andsaid candidate substance.

[0368] In one embodiment of the screening method defined above, thecomplexes formed between the polypeptide and the candidate substance arefurther incubated in the presence of a polyclonal or a monoclonalantibody that specifically binds to the CIDE B protein or to saidfragment or variant thereof

[0369] Various candidate substances or molecules can be assayed forinteraction with a CIDE B polypeptide. These substances or moleculesinclude, without being limited to, natural or synthetic organiccompounds or molecules of biological origin such as polypeptides. Whenthe candidate substance or molecule consists of a polypeptide, thispolypeptide may be the resulting expression product of a phage clonebelonging to a phage-based random peptide library, or alternatively thepolypeptide may be the resulting expression product of a cDNA librarycloned in a vector suitable for performing a two-hybrid screening assay.

[0370] In another embodiment of the present screening method, increasingconcentrations of a monoclonal or polyclonal antibody directed against aCIDE B protein or a fragment or a variant thereof is reacted with theconsidered CIDE B protein or with a fragment or variant thereof,simultaneously or prior to the addition of the candidate substance ormolecule, when performing step c) of said method. By this technique, thedetection and optionally the quantification of the complexes formedbetween the CIDE B protein or the fragment or variant thereof and thesubstance or molecule to be screened allows the one skilled in the artto determine the affinity value of said substance or molecule for saidCIDE B protein or the fragment or variant thereof.

[0371] The invention also pertains to kits useful for performing thehereinbefore described screening method. Preferably, such kits comprisea CIDE B polypeptide or a fragment or a variant thereof and optionallymeans useful to detect the complex formed between the CIDE B polypeptideor its fragment or variant and the candidate substance. In a preferredembodiment the detection means consist in monoclonal or polyclonalantibodies directed against The CIDE B polypeptide or a fragment or avariant thereof.

[0372] 1. Candidate Ligands Obtained from Random Peptide Libraries

[0373] In a particular embodiment of the screening method, the putativeligand is the expression product of a DNA insert contained in a phagevector (Parmley and Smith, 1988). Specifically, random peptide phageslibraries are used. The random DNA inserts encode for peptides of 8 to20 amino acids in length (Oldenburg K. R. et al., 1992; Valadon P., etal., 1996; Lucas A. H., 1994; Westerink M. A. J., 1995; Castagnoli L. etal., 1991). According to this particular embodiment, the recombinantphages expressing a protein that binds to the immobilized CIDE B proteinis retained and the complex formed between the CIDE B protein and therecombinant phage may be subsequently immunoprecipitated by a polyclonalor a monoclonal antibody directed against the CIDE B protein.

[0374] Once the ligand library in recombinant phages has beenconstructed, the phage population is brought into contact with theimmobilized CIDE B protein. Then the preparation of complexes is washedin order to remove the non-specifically bound recombinant phages. Thephages that bind specifically to the CIDE B protein are then eluted by abuffer (acid pH) or immunoprecipitated by the monoclonal antibodyproduced by the hybridoma anti-CIDE B, and this phage population issubsequently amplified by an over-infection of bacteria (for example E.coli). The selection step may be repeated several times, preferably 2-4times, in order to select the more specific recombinant phage clones.The last step consists in characterizing the peptide produced by theselected recombinant phage clones either by expression in infectedbacteria and isolation, expressing the phage insert in anotherhost-vector system, or sequencing the insert contained in the selectedrecombinant phages.

[0375] 2. Candidate Ligands Obtained Through a Two-hybrid ScreeningAssay.

[0376] The yeast two-hybrid system is designed to study protein-proteininteractions in vivo (Fields and Song, 1989), and relies upon the fusionof a bait protein to the DNA binding domain of the yeast Gal4 protein.This technique is also described in the U.S. Pat. No. 5,667,973 and theU.S. Pat. No. 5,283,173 (Fields et al.).

[0377] The general procedure of library screening by the two-hybridassay may be performed as described by Harper et al. (1993) or asdescribed by Cho et al. (1998) or also Fromont-Racine et al. (1997).

[0378] The bait protein or polypeptide consists of a CIDE B polypeptideor a fragment or variant thereof.

[0379] More precisely, the nucleotide sequence encoding the CIDE Bpolypeptide or a fragment or variant thereof is fused to apolynucleotide encoding the DNA binding domain of the GAL4 protein, thefused nucleotide sequence being inserted in a suitable expressionvector, for example pAS2 or pM3.

[0380] Then, a human cDNA library is constructed in a specially designedvector, such that the human cDNA insert is fused to a nucleotidesequence in the vector that encodes the transcriptional domain of theGAL4 protein. Preferably, the vector used is the pACT vector. Thepolypeptides encoded by the nucleotide inserts of the human cDNA libraryare termed “pray” polypeptides.

[0381] A third vector contains a detectable marker gene, such as betagalactosidase gene or CAT gene that is placed under the control of aregulation sequence that is responsive to the binding of a complete Gal4protein containing both the transcriptional activation domain and theDNA binding domain. For example, the vector pG5EC may be used.

[0382] Two different yeast strains are also used. As an illustrative butnon limiting example the two different yeast strains may be thefollowings:

[0383] Y190, the phenotype of which is (MATa, Leu2-3, 112 ura3-12,trp1-901, his3-D200, ade2-101, gal4Dgal180D URA3 GAL-LacZ, LYS GAL-HIS3,cyh^(r));

[0384] Y187, the phenotype of which is (MATa gal4 gal80his3 trp1-901ade2-101 ura3-52 leu2-3, -112 URA3 GAL-lacZmet′), which is the oppositemating type of Y190.

[0385] Briefly, 20 μg of pAS2/CIDE B and 20 μg of pACT-cDNA library areco-transformed into yeast strain Y190. The transformants are selectedfor growth on minimal media lacking histidine, leucine and tryptophan,but containing the histidine stnthesis inhibitor 3-AT (50 mM). Positivecolonies are screened for beta galactosidase by filter lift assay. Thedouble positive colonies (His⁺, beta-gal⁺) are then grown on plateslacking histidine, leucine, but containing tryptophan and cycloheximide(10 mg/ml) to select for loss of pAS2/CIDE B plasmids bu retention ofpACT-cDNA library plasmids. The resulting Y190 strains are mated withYl87 strains expressing CIDE B or non-related control proteins; such ascyclophilin B, lamin, or SNF1, as Gal4 fusions as described by Harper etal. (1993) and by Bram et al. (1993), and screened for betagalactosidase by filter lift assay. Yeast clones that are beta gal-after mating with the control Gal4 fusions are considered falsepositives.

[0386] In another embodiment of the two-hybrid method according to theinvention, interaction between CIDE B or a fragment or variant thereofwith cellular proteins may be assessed using the Matchmaker Two HybridSystem 2 (Catalog No. K1604-1, Clontech).). As described in the manualaccompanying the Matchmaker Two Hybrid System 2 (Catalog No. K1604-1,Clontech), the disclosure of which is incorporated herein by reference,nucleic acids encoding the RBP-7 protein or a portion thereof, areinserted into an expression vector such that they are in frame with DNAencoding the DNA binding domain of the yeast transcriptional activatorGAL4. A desired cDNA, preferably human cDNA, is inserted into a secondexpression vector such that they are in frame with DNA encoding theactivation domain of GAL4. The two expression plasmids are transformedinto yeast and the yeast are plated on selection medium which selectsfor expression of selectable markers on each of the expression vectorsas well as GAL4 dependent expression of the HIS3 gene. Transformantscapable of growing on medium lacking histidine are screened for GAL4dependent lacZ expression. Those cells which are positive in both thehistidine selection and the lacZ assay contain interaction between CIDEB and the protein or peptide encoded by the initially selected cDNAinsert.

[0387] 3. Candidate Ligands Obtained by Affinity Chromatography.

[0388] Proteins or other molecules interacting with the CIDE B protein,or a fragment thereof can also be found using affinity columns whichcontain the CIDE B protein, or a fragment thereof. The CIDE B protein,or a fragment thereof, may be attached to the column using conventionaltechniques including chemical coupling to a suitable column matrix suchas agarose, Affi Gel®, or other matrices familiar to those of skill inart. In some embodiments of this method, the affinity column containschimeric proteins in which the CIDE B protein, or a fragment thereof, isfused to glutathion S transferase (GST). A mixture of cellular proteinsor pool of expressed proteins as described above is applied to theaffinity column. Proteins or other molecules interacting with the CIDE Bprotein, or a fragment thereof, attached to the column can then beisolated and analyzed on 2-D electrophoresis gel as described inRamunsen et al. (1997), the disclosure of which is incorporated byreference. Alternatively, the proteins retained on the affinity columncan be purified by electrophoresis based methods and sequenced. The samemethod can be used to isolate antibodies, to screen phage displayproducts, or to screen phage display human antibodies.

[0389] 4. Candidate Ligands Obtained by Optical Biosensor Methods

[0390] Proteins interacting with the CIDE B protein, or a fragmentcomprising a contiguous span of at least 6 amino acids, preferably atleast 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30,40, 50, or 100 amino acids of SEQ ID No 3, wherein said contiguous spanincludes at least 1, 2, 3, 5 or 10 of the amino acid positions 7-11,18-29, 47, 55-63, 70, 103-104, 111-115, 124, 134, 169-173, 181-185, and203-219, can also be screened by using an Optical Biosensor as describedin Edwards and Leatherbarrow (1997) and also in Szabo et al. (1995), thedisclosure of which is incorporated by reference. This technique permitsthe detection of interactions between molecules in real time, withoutthe need of labeled molecules. This technique is based on the surfaceplasmon resonance (SPR) phenomenon. Briefly, the candidate ligandmolecule to be tested is attached to a surface (such as a carboxymethyldextran matrix). A light beam is directed towards the side of thesurface that does not contain the sample to be tested and is reflectedby said surface. The SPR phenomenon causes a decrease in the intensityof the reflected light with a specific association of angle andwavelength. The binding of candidate ligand molecules cause a change inthe refraction index on the surface, which change is detected as achange in the SPR signal. For screening of candidate ligand molecules orsubstances that are able to interact with the CIDE B protein, or afragment thereof, the CIDE B protein, or a fragment thereof, isimmobilized onto a surface. This surface comprises one side of a cellthrough which flows the candidate molecule to be assayed. The binding ofthe candidate molecule on the CIDE B protein, or a fragment thereof, isdetected as a change of the SPR signal. The candidate molecules testedmay be proteins, peptides, carbohydrates, lipids, or small moleculesgenerated by combinatorial chemistry. This technique may also beperformed by immobilizing eukaryotic or prokaryotic cells or lipidvesicles exhibiting an endogenous or a recombinantly expressed CIDE Bprotein at their surface.

[0391] The main advantage of the method is that it allows thedetermination of the association rate between the CIDE B protein andmolecules interacting with the CIDE B protein. It is thus possible toselect specifically ligand molecules interacting with the CIDE Bprotein, or a fragment thereof, through strong or conversely weakassociation constants.

[0392] Method for Screening Ligands that Modulate the Expression of theCIDE B Gene.

[0393] Another subject of the present invention is a method forscreening molecules that modulate the expression of the CIDE B protein.Such a screening method comprises the steps of:

[0394] a) cultivating a prokaryotic or an eukaryotic cell that has beentransfected with a nucleotide sequence encoding the CIDE B protein or avariant or a fragment thereof, placed under the control of its ownpromoter;

[0395] b) bringing into contact the cultivated cell with a molecule tobe tested;

[0396] c) quantifying the expression of the CIDE B protein or a variantor a fragment thereof.

[0397] Using DNA recombination techniques well known by the one skill inthe art, the CIDE B protein encoding DNA sequence is inserted into anexpression vector, downstream from its promoter sequence. As anillustrative example, the promoter sequence of the CIDE B gene iscontained in the 5′ regulatory region of CIDE B.

[0398] The quantification of the expression of the CIDE B protein may berealized either at the mRNA level or at the protein level. In the lattercase, polyclonal or monoclonal antibodies may be used to quantify theamounts of the CIDE B protein that have been produced, for example in anELISA or a RIA assay.

[0399] In a preferred embodiment, the quantification of the CIDE B mRNAis realized by a quantitative PCR amplification of the cDNA obtained bya reverse transcription of the total mRNA of the cultivated CIDEB-transfected host cell, using a pair of primers specific for CIDE B.

[0400] The present invention also concerns a method for screeningsubstances or molecules that are able to increase, or in contrast todecrease, the level of expression of the CIDE B gene. Such a method mayallow the one skilled in the art to select substances exerting aregulating effect on the expression level of the CIDE B gene and whichmay be useful as active ingredients included in pharmaceuticalcompositions for treating patients suffering from deficiencies in theregulation of expression of the CIDE B gene.

[0401] Thus, is also part of the present invention a method for thescreening of a candidate substance or molecule, said method comprisingthe following steps:

[0402] a) providing a recombinant cell host containing a nucleic acid,wherein said nucleic acid comprises a nucleotide sequence selected fromthe group consisting of SEQ ID Nos 1 and 2;

[0403] b) obtaining a candidate substance, and

[0404] c) determining the ability of the candidate substance to modulatethe expression levels of the nucleotide sequences selected from thegroup consisting of SEQ ID Nos 1 and 2.

[0405] The invention also pertains to kits useful for performing thehereinbefore described screening method. Preferably, such kits comprisea recombinant vector that allows the expression of a nucleotide sequenceselected from the group consisting of SEQ ID Nos 1 and 2 oralternatively a recombinant cell host containing such a recombinantvector.

[0406] In another embodiment of a method for screening of a candidatesubstance or molecule that modulated the expression of the CIDE B gene,this method comprises the following steps:

[0407] a) providing a recombinant cell host containing a nucleic acid,wherein said nucleic acid comprises the 5′ regulatory region of CIDE Bor a biologically active fragment or variant thereof located upstream apolynucleotide encoding a detectable protein;

[0408] b) obtaining a candidate substance, and

[0409] c) determining the ability of the candidate substance to modulatethe expression levels of the polynucleotide encoding the detectableprotein.

[0410] Among the preferred polynucleotides encoding a detectableprotein, there may be cited polynucleotides encoding beta galactosidase,green fluorescent protein (GFP) and chloramphenicol acetyl transferase(CAT).

[0411] The invention also pertains to kits useful for performing thehereinbefore described screening method. Preferably, such kits comprisea recombinant vector comprising the 5′ regulatory region of CIDE B or abiologically active fragment or variant thereof located upstream andoperably linked to a polynucleotide encoding a detectable protein or theCIDE B protein or a fragment or a variant thereof.

[0412] For the design of suitable recombinant vectors useful forperforming the screening methods described above, it will be referred tothe section of the present specification wherein the preferredrecombinant vectors of the invention are detailed.

[0413] Expression levels and patterns of CIDE B may be analyzed bysolution hybridization with long probes as described in InternationalPatent Application No. WO 97/05277, the entire contents of which areincorporated herein by reference. Briefly, the CIDE B cDNA or the CIDE Bgenomic DNA described above, or fragments thereof, is inserted at acloning site immediately downstream of a bacteriophage (T3, T7 or SP6)RNA polymerase promoter to produce antisense RNA. Preferably, the CIDE Binsert comprises at least 100 or more consecutive nucleotides of thegenomic DNA sequence or the cDNA sequences, particularly thosecomprising at least one of SEQ ID Nos 15-18 or those encoding mutatedCIDE B. The plasmid is linearized and transcribed in the presence ofribonucleotides comprising modified ribonucleotides (i.e. biotin-UTP andDIG-UTP). An excess of this doubly labeled RNA is hybridized in solutionwith mRNA isolated from cells or tissues of interest. The hybridizationsare performed under standard stringent conditions (40-50° C. for 16hours in an 80% formamide, 0.4 M NaCl buffer, pH 7-8). The unhybridizedprobe is removed by digestion with ribonucleases specific forsingle-stranded RNA (i.e. RNases CL3, T1, Phy M, U2 or A). The presenceof the biotin-UTP modification enables capture of the hybrid on amicrotitration plate coated with streptavidin. The presence of the DIGmodification enables the hybrid to be detected and quantified by ELISAusing an anti-DIG antibody coupled to alkaline phosphatase.

Methods for Inhibiting the Expression of a CIDE B Gene

[0414] Other therapeutic compositions according to the present inventioncomprise advantageously an oligonucleotide fragment of the nucleicsequence of CIDE B as an antisense tool that inhibits the expression ofthe corresponding CIDE B gene. Preferred methods using antisensepolynucleotide according to the present invention are the proceduresdescribed by Sczakiel et al. (1995).

[0415] Preferably, the antisense tools are chosen among thepolynucleotides (15-200 bp long) that are complementary to the 5′end ofthe CIDE B mRNA. In another embodiment, a combination of differentantisense polynucleotides complementary to different parts of thedesired targeted gene are used.

[0416] Preferred antisense polynucleotides according to the presentinvention are complementary to a sequence of the mRNAs of CIDE B thatcontains the translation initiation codon ATG.

[0417] The antisense nucleic acid molecules to be used in gene therapymay be either DNA or RNA sequences. They comprise a nucleotide sequencecomplementary to the targeted sequence of the CIDE B genomic DNA, thesequence of which can be determined using one of the detection methodsof the present invention. In a preferred embodiment, the antisenseoligonucleotide are able to hybridize with at least one of the splicingsites of the targeted CIDE B gene, or with the 3′UTR of the 5′UTR. Theantisense nucleic acids should have a length and melting temperaturesufficient to permit formation of an intracellular duplex havingsufficient stability to inhibit the expression of the CIDE B mRNA in theduplex. Strategies for designing antisense nucleic acids suitable foruse in gene therapy are disclosed in Green et al., (1986) and Izant andWeintraub, (1984).

[0418] In some strategies, antisense molecules are obtained by reversingthe orientation of the CIDE B coding region with respect to a promoterso as to transcribe the opposite strand from that which is normallytranscribed in the cell. The antisense molecules may be transcribedusing in vitro transcription systems such as those which employ T7 orSP6 polymerase to generate the transcript. Another approach involvestranscription of CIDE B antisense nucleic acids in vivo by operablylinking DNA containing the antisense sequence to a promoter in asuitable expression vector.

[0419] Alternatively, suitable antisense strategies are those describedby Rossi et al. (1991), in the International Applications Nos. WO94/23026, WO 95/04141, WO 92/18522 and in the European PatentApplication No. EP 0 572 287 A2

[0420] An alternative to the antisense technology that is used accordingto the present invention consists in using ribozymes that will bind to atarget sequence via their complementary polynucleotide tail and thatwill cleave the corresponding RNA by hydrolyzing its target site (namely<<hammerhead ribozymes>>. Briefly, the simplified cycle of a hammerheadribozyme consists of (1) sequence specific binding to the target RNA viacomplementary antisense sequences; (2) site-specific hydrolysis of thecleavable motif of the target strand; and (3) release of cleavageproducts, which gives rise to another catalytic cycle. Indeed, the useof long-chain antisense polynucleotide (at least 30 bases long) orribozymes with long antisense arms are advantageous. A preferreddelivery system for antisense ribozyme is achieved by covalently linkingthese antisense ribozymes to lipophilic groups or to use liposomes as aconvenient vector. Preferred antisense ribozymes according to thepresent invention are prepared as described by Sczakiel et al. (1995).

[0421] Throughout this application, various publications, patents andpublished patent applications are cited. The disclosures of thesepublications, patents and published patent specification referenced inthis application are hereby incorporated by reference into the presentdisclosure to more fully describe the sate of the art to which thisinvention pertains.

EXAMPLES Example 1 Identification of Biallelic Markers—DNA Extraction

[0422] Donors were unrelated and healthy. They presented a sufficientdiversity for being representative of a French heterogeneous population.The DNA from 100 individuals was extracted and tested for the detectionof the biallelic markers.

[0423] 30 ml of peripheral venous blood were taken from each donor inthe presence of EDTA. Cells (pellet) were collected after centrifugationfor 10 minutes at 2000 rpm. Red cells were lysed by a lysis solution (50ml final volume: 10 mM Tris pH7.6; 5 mM MgCl₂; 10 mM NaCl). The solutionwas centrifuged (10 minutes, 2000 rpm) as many times as necessary toeliminate the residual red cells present in the supernatant, afterresuspension of the pellet in the lysis solution.

[0424] The pellet of white cells was lysed overnight at 42° C. with 3.7ml of lysis solution composed of:

[0425] 3 ml TE 10-2 (Tris-HCl 10 mM, EDTA 2 mM)NaCl 0 4 M

[0426] 200 μl SDS 10%

[0427] 500 μl K-proteinase (2 mg K-proteinase in TE 10-2/NaCl 0.4 M).

[0428] For the extraction of proteins, 1 ml saturated NaCl (6M) (1/3.5v/v) was added. After vigorous agitation, the solution was centrifugedfor 20 minutes at 10000 rpm.

[0429] For the precipitation of DNA, 2 to 3 volumes of 100% ethanol wereadded to the previous supernatant, and the solution was centrifuged for30 minutes at 2000 rpm. The DNA solution was rinsed three times with 70%ethanol to eliminate salts, and centrifuged for 20 minutes at 2000 rpm.The pellet was dried at 37° C., and resuspended in 1 ml TE 10-1 or 1 mlwater. The DNA concentration was evaluated by measuring the OD at 260 nm(1 unit OD=50 μg/ml DNA).

[0430] To determine the presence of proteins in the DNA solution, the OD260/OD 280 ratio was determined. Only DNA preparations having a OD260/OD 280 ratio between 1.8 and 2 were used in the subsequent examplesdescribed below.

[0431] The pool was constituted by mixing equivalent quantities of DNAfrom each individual.

Example 2 Identification of Biallelic Markers: Amplification of GenomicDNA by PCR

[0432] The amplification of specific genomic sequences of the DNAsamples of example 1 was carried out on the pool of DNA obtainedpreviously. In addition, 50 individual samples were similarly amplified.

[0433] PCR assays were performed using the following protocol: Finalvolume 25 μl DNA 2 ng/μl MgCl₂ 2 mM dNTP (each) 200 μM primer (each) 2.9ng/μl Ampli Taq Gold DNA polymerase 0.05 unit/ml PCR buffer (10x = 0.1 MTrisHCl pH 8.3 0.5 M KCl) 1 x

[0434] Each pair of first primers was designed using the sequenceinformation of the CIDE B gene disclosed herein and the OSP software(Hillier & Green, 1991). This first pair of primers was about 20nucleotides in length and had the sequences disclosed in Table 1 in thecolumns labeled PU and RP. TABLE 1 Position Position range ofComplmentary range of the Position range of position range of ampliconin Primer amplification primer Primer amplification primer Amplicon SEQID 1 name in SEQ ID No 1 name in SEQ ID No 1 12-73 6704 7169 B(12- 67046723 C(12- 7152 7169 73) 73) 12-74 9538 9988 B(12- 9538 9557 C(12- 99709988 74) 74)

[0435] Preferably, the primers contained a common oligonucleotide tailupstream of the specific bases targeted for amplification which wasuseful for sequencing.

[0436] Primers PU contain the following additional PU 5′ sequence:

[0437] TGTAAAACGACGGCCAGT; primers RP contain the following RP 5′sequence:

[0438] CAGGAAACAGCTATGACC. The primer containing the additional PU 5′sequence is listed in SEQ ID No 4. The primer containing the additionalRP 5′ sequence is listed in SEQ ID No 5.

[0439] The synthesis of these primers was performed following thephosphoramidite method, on a GENSET UFPS 24.1 synthesizer.

[0440] DNA amplification was performed on a Genius II thermocycler.After heating at 95° C. for 10 min, 40 cycles were performed. Each cyclecomprised: 30 sec at 95° C., 54° C. for 1 min, and 30 sec at 72° C. Forfinal elongation, 10 min at 72° C. ended the amplification. Thequantities of the amplification products obtained were determined on96-well microtiter plates, using a fluorometer and Picogreen asintercalant agent (Molecular Probes).

Example 3 Identification of Biallelic Markers—Sequencing of AmplifiedGenomic DNA and Identification of Polymorphisms

[0441] The sequencing of the amplified DNA obtained in example 2 wascarried out on ABI 377 sequencers. The sequences of the amplificationproducts were determined using automated dideoxy terminator sequencingreactions with a dye terminator cycle sequencing protocol. The productsof the sequencing reactions were run on sequencing gels and thesequences were determined using gel image analysis (ABI Prism DNASequencing Analysis software (2.1.2 version)).

[0442] The sequence data were further evaluated to detect the presenceof biallelic markers within the amplified fragments. The polymorphismsearch was based on the presence of superimposed peaks in theelectrophoresis pattern resulting from different bases occurring at thesame position as described previously.

[0443] In the 2 fragments of amplification, 2 biallelic markers weredetected. The localization of these biallelic markers are as shown inTable 2. TABLE 2 Marker Localization in Polymorphism BM position inAmplicon Name CIDE B gene allele1 allele2 SEQ ID No 1 12-73 12-73-49 3′regulatory region C T 7123 12-74 12-74-38 3′ regulatory region C T 9574

[0444] TABLE 3 Position range of Marker Name probes in SEQ ID No 1Probes 12-73-49 7100 7146 P(12-73-49) 12-74-38 9551 9597 P(12-74-38)

Example 4 Validation of the Polymorphisms Through Microsequencing

[0445] The biallelic markers identified in example 3 were furtherconfirmed and their respective frequencies were determined throughmicrosequencing. Microsequencing was carried out for each individual DNAsample described in Example 1.

[0446] Amplification from genomic DNA of individuals was performed byPCR as described above for the detection of the biallelic markers withthe same set of PCR primers (Table 1).

[0447] The preferred primers used in microsequencing were about 19nucleotides in length and hybridized just upstream of the consideredpolymorphic base. According to the invention, the primers used inmicrosequencing are detailed in Table 4. TABLE 4 Position range ofComplementary position microsequencing range of Marker primer mis 1 inmicrosequencing primer Name Mis. 1 SEQ ID No 1 Mis. 2 mis. 2 in SEQ IDNo 1 12-73-49 D(12-73-49) 7104 7122 E(12-73-49) 7124 7142 12-74-38S(12-74-38) 9555 9573 E(12-74-38) 9575 9593

[0448] Mis 1 and Mis 2 respectively refer to microsequencing primerswhich hybridized with the non-coding strand of the CIDE B gene or withthe coding strand of the CIDE B gene.

[0449] The microsequencing reaction was performed as follows:

[0450] After purification of the amplification products, themicrosequencing reaction mixture was prepared by adding, in a 20 μlfinal volume: 10 pmol microsequencing oligonucleotide, 1 UThermosequenase (Amersham E79000G), 1.25 μl Thermosequenase buffer (260mM Tris HCl pH 9.5, 65 mM MgCl₂), and the two appropriate fluorescentddNTPs (Perkin Elmer, Dye Terminator Set 401095) complementary to thenucleotides at the polymorphic site of each biallelic marker tested,following the manufacturer's recommendations. After 4 minutes at 94° C.,20 PCR cycles of sec at 55° C., 5 sec at 72° C., and 10 sec at 94° C.were carried out in a Tetrad PTC-225 (MJ Research). The unincorporateddye terminators were then removed by ethanol precipitation. Samples werefinally resuspended in formamide-EDTA loading buffer and heated for 2min at 95° C. before being loaded on a polyacrylamide sequencing gel.The data were collected by an ABI PRISM 377 DNA sequencer and processedusing the GENESCAN software (Perkin Elmer).

[0451] Following gel analysis, data were automatically processed withsoftware that allows the determination of the alleles of biallelicmarkers present in each amplified fragment.

[0452] The software evaluates such factors as whether the intensities ofthe signals resulting from the above microsequencing procedures areweak, normal, or saturated, or whether the signals are ambiguous. Inaddition, the software identifies significant peaks (according to shapeand height criteria). Among the significant peaks, peaks correspondingto the targeted site are identified based on their position. When twosignificant peaks are detected for the same position, each sample iscategorized classification as homozygous or heterozygous type based onthe height ratio.

Example 5 Preparation of Antibody Compositions to the CIDE B Protein

[0453] Substantially pure protein or polypeptide is isolated fromtransfected or transformed cells containing an expression vectorencoding the CIDE B protein or a portion thereof. The concentration ofprotein in the final preparation is adjusted, for example, byconcentration on an Amicon filter device, to the level of a fewmicrograms/ml. Monoclonal or polyclonal antibody to the protein can thenbe prepared as follows:

[0454] A. Monoclonal Antibody Production by Hybridoma Fusion

[0455] Monoclonal antibody to epitopes in the CIDE B protein or aportion thereof can be prepared from murine hybridomas according to theclassical method of Kohler, G. and Milstein, C., (1975) or derivativemethods thereof. Also see Harlow, E., and D. Lane. 1988.

[0456] Briefly, a mouse is repetitively inoculated with a few microgramsof the CIDE B protein or a portion thereof over a period of a few weeks.The mouse is then sacrificed, and the antibody producing cells of thespleen isolated. The spleen cells are fused by means of polyethyleneglycol with mouse myeloma cells, and the excess unfused cells destroyedby growth of the system on selective media comprising aminopterin (HATmedia). The successfully fused cells are diluted and aliquots of thedilution placed in wells of a microtiter plate where growth of theculture is continued. Antibody-producing clones are identified bydetection of antibody in the supernatant fluid of the wells byimmunoassay procedures, such as ELISA, as originally described byEngvall, (1980), and derivative methods thereof. Selected positiveclones can be expanded and their monoclonal antibody product harvestedfor use. Detailed procedures for monoclonal antibody production aredescribed in Davis, L. et al. Basic Methods in Molecular BiologyElsevier, New York. Section 21-2.

[0457] B. Polyclonal Antibody Production by Immunization

[0458] Polyclonal antiserum containing antibodies to heterogeneousepitopes in the CIDE B protein or a portion thereof can be prepared byimmunizing suitable non-human animal with the CIDE B protein or aportion thereof, which can be unmodified or modified to enhanceimmmunogenicity. A suitable non-human animal is preferably a non-humanmammal is selected, usually a mouse, rat, rabbit, goat, or horse.Alternatively, a crude preparation which has been enriched for CIDE Bconcentration can be used to generate antibodies. Such proteins,fragments or preparations are introduced into the non-human mammal inthe presence of an appropriate adjuvant (e.g. aluminum hydroxide, RIBI,etc.) which is known in the art. In addition the protein, fragment orpreparation can be pretreated with an agent which will increaseantigenicity, such agents are known in the art and include, for example,methylated bovine serum albumin (mBSA), bovine serum albumin (BSA),Hepatitis B surface antigen, and keyhole limpet hemocyanin (KLH). Serumfrom the immunized animal is collected, treated and tested according toknown procedures. If the serum contains polyclonal antibodies toundesired epitopes, the polyclonal antibodies can be purified byimmunoaffinity chromatography.

[0459] Effective polyclonal antibody production is affected by manyfactors related both to the antigen and the host species. Also, hostanimals vary in response to site of inoculations and dose, with bothinadequate or excessive doses of antigen resulting in low titerantisera. Small doses (ng level) of antigen administered at multipleintradermal sites appears to be most reliable. Techniques for producingand processing polyclonal antisera are known in the art, see forexample, Mayer and Walker (1987). An effective immunization protocol forrabbits can be found in Vaitukaitis, J. et al. (1971).

[0460] Booster injections can be given at regular intervals, andantiserum harvested when antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall. See, forexample, Ouchterlony, O. et al., (1973). Plateau concentration ofantibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12μM). Afinity of the antisera for the antigen is determined by preparingcompetitive binding curves, as described, for example, by Fisher, D.,(1980).

[0461] Antibody preparations prepared according to either the monoclonalor the polyclonal protocol are useful in quantitative immunoassays whichdetermine concentrations of antigen-bearing substances in biologicalsamples; they are also used semi-quantitatively or qualitatively toidentify the presence of antigen in a biological sample. The antibodiesmay also be used in therapeutic compositions for killing cellsexpressing the protein or reducing the levels of the protein in thebody.

[0462] While the preferred embodiment of the invention has beenillustrated and described, it will be appreciated that various changescan be made therein by the one skilled in the art without departing fromthe spirit and scope of the invention.

REFERENCES

[0463] Altschul et al., 1990, J. Mol. Biol. 215(3):403-410/Altschul etal., 1993, Nature Genetics 3:266-272/Altschul et al., 1997, Nuc. AcidsRes. 25:3389-3402/Ausubel et al. (1989)Current Protocols in MolecularBiology, Green Publishing Associates and Wiley Interscience, N.Y./BatesG P et al., 1997a, Hum. Mol. Genet., 6(10):1633-1637./Bates G P et al.,1997b, Molecular Medicine today, November 1997, 508:515./Beaucage etal., Tetrahedron Lett 1981, 22:1859-1862/Bram R J et al., 1993, Mol.Cell Biol., 13:4760-4769/Brown E L, et al., Methods Enzymol1979;68:109-151/Burright et al., 1997, Brain Pathology,7:965-977./Castagnoli L. et al. (Felici F.), 1991, J. Mol. Biol.,222:301-310/Chai H. et al. (1993) Biotechnol. Appl.Biochem.18:259-273./Chee et al. (1996) Science. 274:610-614./Chen andKwok Nucleic Acids Research 25:347-353 1997/Chen et al. Proc. Natl.Acad. Sci. USA 94/20 10756-10761, 1997/Cho R X et al., 1998, Proc. Natl.Acad. Sci. USA, 95(7):3752-3757./Coles R, et al, Hum Mol Genet1998;7(5):791-800/Compton J. (1991) Nature. 350(6313):91-92./Davies S W,et al. Cell 1997;90(3):537-48/Edwards et Leatherbarrow, AnalyticalBiochemistry, 246, 1-6 (1997)/Engvall, E., Meth. Enzymol. 70:419(1980)/Feldman and Steg, 1996, Medecine/Sciences, synthese,12:47-55/Fields and Song, 1989, Nature, 340:245-246/Fisher, D., Chap. 42in: Manual of Clinical Immunology, 2d Ed. (Rose and Friedman, Eds.)Amer. Soc. For Microbiol., Washington, D.C. (1980) /Flotte et al., 1992,Am. J. Respir. Cell Mol. Biol., 7:349-356./Fodor et al. (1991) Science251:767-777./Fromont-Racine M. et al., 1997, Nature Genetics,16(3):277-282./Fuller S. A. et al. (1996) Immunology in CurrentProtocols in Molecular Biology, Ausubel et al.Eds, John Wiley & Sons,Inc., USA./Geysen H. Mario et al. 1984. Proc. Natl. Acad. Sci. U.S.A.81:3998-4002/Gonnet et al., 1992, Science 256.1443-1445/Green et al.,Ann. Rev. Biochem. 55:569-597 (1986)/Grompe, M. (1993) Nature Genetics.5:111-117./Grompe, M. et al. (1989)Proc. Natl. Acad. Sci. U.S.A.86:5855-5892./Guatelli J C et al. Proc. Natl. Acad. Sci. USA.35:273-286./Hacia J G, et al., Nat Genet 1996;14(4):441-447/Haff L. A.and Smirnov I. P. (1997) Genome Research, 7:378-388./Hames B. D. andHiggins S. J. (1985) Nucleic Acid Hybridization: A Practical Approach.Hames and Higgins Ed., IRL Press, Oxford./Harju L, et al., Clin Chem1993;39(11Pt 1):2282-2287/Harlow, E., and D. Lane. 1988. Antibodies ALaboratory Manual. Cold Spring Harbor Laboratory. pp. 53-242/Harper J Wet al., 1993, Cell, 75:805-816/Henikoff and Henikoff, 1993, Proteins17:49-61/Higgins et al., 1996, Methods Enzymol. 266:383-402/Hillier L.and Green P. Methods Appl, 1991, 1:124-8./Huang L. et al. (1996) CancerRes 56(5):1137-1141./Huygen et al. (1996) Nature Medicine.2(8):893-898./Inohara N. et al., 1998, The EMBO J.,17(9),:2526-2533./Izant J G, Weintraub H, Cell 1984Apr;36(4):1007-15/Julan et al. 1992, J. Gen. Virol., 73:3251-3255./Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA87:2267-2268/Kohler, G. and Milstein, C., Nature 256:495 (1975)/Kozal MJ, et al., Nat Med 1996;2(7):753-759/Landegren U. et al. (1998) GenomeResearch, 8:769-776./Leger O J, et al., 1997, Hum Antibodies, 8(1):3-16/Lenhard T. et al. (1996) Gene. 169:187-190./Lin M W et al., 1997,Hum. Genet., 99(3):417-420./Livak et al., Nature Genetics, 9:341-342,1995/Livak K J, Hainer J W, Hum Mutat 1994;3(4):379-385/Lucas A. H.,1994, In : Development and Clinical Uses of Haempophilus bConjugate;/Mackey K, et al., 1998, Mol Biotechnol, 9(1):1-5/MangiariniL, et al. Nat Genet Feburary 1997;15(2):197-200/Mansour S L et al.,1988, Nature, 336:348-352./Marshall R. L. et al. (1994) PCR Methods andApplications. 4:80-84./Martineau P, et al., 1998, J. Mol Biol,280(1):117-127/McLaughlin B A et al., 1996, Am. J Hum. Genet.,59:561-569./Muzyczka et al., 1992, Curr. Topics in Micro. and Immunol.,158:97-129. /Narang S A, et al., Methods Enzymol 1979;68:90-98/Neda etal., 1991, J Biol. Chem., 266:14143-14146./Nickerson D. A. et al. (1990)Proc. Natl. Acad. Sci. U.S.A. 87:8923-8927./Nyren P, et al., AnalBiochem 1993;208(1):171-175/O'Reilly et al. (1992) BaculovirusExpression Vectors: A Laboratory Manual. W. H. Freeman and Co., NewYork./Ohno et al. (1994) Science. 265:781-784./Oldenburg K. R. et al.,1992, Proc. Natl. Acad. Sci., 89:5393-5397./Orita et al. (1989) Proc.Natl. Acad. Sci. U.S.A. 86:2776-2770./Ouchterlony, O. et al., Chap. 19in: Handbook of Experimental Immunology D. Wier (ed) Blackwell(1973)/Parmley and Smith, Gene, 1988, 73:305-318/Pastinen et al., GenomeResearch 1997; 7:606-614/Pearson and Lipman, 1988, Proc. Natl. Acad.Sci. USA 85(8):2444-2448/Porath et al., Nature December 18,1975;258(5536):598-9/Ramunsen et al., 1997, Electrophoresis,18:588-598./Reimann K A, et al, 1997, AIDS Res Hum Retroviruses.13(11):933-943/Ridder R, et al., 1995, Biotechnology (NY)13(3):255-260/Rossi et al., Pharmacol. Ther. 50:245-254, (1991)/Roth J.A. et al., 1996, Nature Medicine, 2(9):985-991/Rougeo, C. et al.,. Eur.J Biochem. 219 (3):765-773, 1994/Roux et al., 1989, Proc. Natl. Acad.Sci. USA, 86:9079-9083./Sambrook, J., et al.. (1 989) Molecular Cloning:A Laboratory Manual. 2ed. Cold Spring Harbor Laboratory, Cold SpringHarbor, New York./Samson M, et al. (1996) Nature,382(6593):722-725./Samulski et al., 1989, J. Virol.,63:3822-3828./Sanchez-Pescador R. (1988) J. Clin. Microbiol.26(10):1934-1938./Sandou et al., 1994, Science, 265:1875-1878./Schwartzand Dayhoff, eds., 1978, Matrices for Detecting Distance Relationships:Atlas of Protein Sequence and Structure, Washington: National BiomedicalResearch Foundation/Sczakiel G. et al., 1995, Trends Microbiol., 1995,3(6):213-217/Sheffield, V. C. et al. (1991) Proc. Natl. Acad. Sci.U.S.A. 49:699-706./Shoemaker D D, et al., Nat Genet1996;14(4):450-456/Smith et al. (1983) Mol. Cell. Biol.3:2156-2165./Stryer, L., Biochemistry, 4th edition, 1995/Syvanen A C,Clin Chim Acta 1994;226(2):225-236/Szabo A. et al. Curr Opin Struct Biol5, 699-705 (1995)/Tacson et al. (1996) Nature Medicine.2(8):888-892./Thompson et al., 1994, Nucleic Acids Res. 22(2):46734680/Tyagi et al. (1998) Nature Biotechnology. 16:49-53./Urdea M. S.(1988) Nucleic Acids Research. 11:4937-4957. /Urdea M. S. et al.(1991)Nucleic Acids Symp. Ser. 24:197-200./Vaitukaitis, J. et al. J. Clin.Endocrinol. Metab. 33:988-991(1971)/Valadon P., et al., 1996, J.Mol.Biol.,261:11-22./Vaughan T J, et al., 1996, Nat Biotechnol. 14(3):309-314/Vlasak R. et al. (1983) Eur. J. Biochem. 135:123-126./Wabiko etal. (1986) DNA.5(4):305-314./Walker et al. (1996) Clin. Chem.42:9-13./Wanker E E, Mangiarini L, Bates G P Cell1997;90(3):537-48/Westerink M. A. J., 1995, Proc. Natl. Acad. Sci.,92:4021-4025/White, M. B. et al. (1992) Genomics. 12:301-306./White, M.B. et al. (1997) Genomics. 12:301-306.

SEQUENCE LISTING FREE TEXT

[0464] The following free text appears in the accompanying SequenceListing:

[0465] 5′ regulatory region

[0466] 3′ regulatory region

[0467] polymorphic base or

[0468] complement

[0469] probe

[0470] homology with 5′ EST in ref

[0471] sequencing oligonucleotide Primer

1 5 1 10961 DNA Homo sapiens misc_feature 1..2802 5′ regulatory region 1ggctgtgcct gtggcccgcg aagtcttcca gctcagcagt gtctcgttcc ctgggggacg 60gtagcagacc gacatccttc tgggcctaca ggtgggtgga aggcgccaca gagggcgttc 120aggggtgggg caaaacccca cctgtgatgc ccgatgagaa ggtgccatgc ctcctcctta 180cttccctgca gcctgcctct tttctgcctg ggagtcctga cttccacgag gacccagacc 240ccacctcaaa cacaactcct tcttggaacc cagatcccct gctcccagtc agttgacctg 300ccccactcct ggcctccttc ccagagctca gtggtaaaca ggcataaagt cctcacccca 360ggctgtcact atctctacca ccactcctct agtctggccc ctccttcttt gtccagcccc 420attctagcac atctgggcaa aactggatgg tggggtgtaa agggacgtgc acagatctac 480ttaccaagct gggagcaagc aggattgggg gcctggagaa gctgaaaggt taagcagcag 540taggcgaggt gcctactcct gtcctgtgcc tatcacattt gcagagggta agacaagaat 600ggggagggac aagatgaggc tcatcctggg cctctcctgg ggcccctccc tcaccctgct 660ccttcccatc accaacgcac ccaggcgtcc aggcatgtga gagcctgcct tgccaggaaa 720cagcacaagc tgaagtgggg cagtaagatt ccctggtggt ggaaggaaat aggaggactc 780tgctgaatcc tggtcctgct tctgttctca tccctccccc agctctggcc ccaatcctcc 840cagcccacct tctgcagtgt gtatgttgcc tggtctctct ggcctgcaga ggtgacccaa 900acaactcagt cccccacctg ccttatcctt gcctgtttcc agtctcctgg tccggctgag 960cccctggcct cctacctcct cccacctctt gcctcaggcc ctgccccagt cactccaccc 1020tcagggctct caccgggctg tcaatgacag cttttccatg taaggcatgg tgctaggttc 1080caggaggaac aggggatgca tggaggcata atggttaggg agtcatgaca cacaaccatg 1140aagaggcccc attaccaggc tgcaccagga tacaagacaa gaaaggaaag gatgagtagg 1200gacatactaa gaagcagccc tctcctcttg gaaaagtcaa gcaaaacttg gaaatgaaga 1260gatttgaggg gggcctggca gatggataga gctgggggaa gggaaaagaa aggcctctgc 1320tcaagtaaca gccagaactt gaggttgctt ggggagggca ccaggagcac tgtcttagtt 1380tgggttcttc caaagcagag cttgagctaa gggcttgggt acaggtgatc ctgtattctt 1440gagctaaggg cttgggtaca ggtgatcctg tatttgggag gttaactcag gaagtgaggg 1500cataaggtaa aacaagagag aggaaagcca ttaagagtat gttaagtccc ttcagtaggc 1560cttgggaacc tctgagaaaa gtatagattg cccaagacaa aagactggca gggtgatcag 1620tccaaagcat ttattaggag aatgtactta atgagtgggc tacagcgtat cctcacaaca 1680gacagtgaga gagagttgtt ctacctgggt atatccaaaa caagggggtc agggtatgga 1740gtttacgagg gttcaaggta tttggttcag ggccagggcc agtttttttc agtgttttgg 1800gcaacaacct gaataccttt tcaaggctct ggcttgggct caagcctgca ggggaaatgc 1860aactggccag gtcacagggc aatcaagtta ctctgtgttt ctttgtcagg acacagaaaa 1920aaagtgggga agctggggga ccctacaagg atccttggca ggaaagcagg gattgtgttc 1980atttgagggt ttcactgtca gtgagagtct cagcttccat gcaactgtcc atcacggctg 2040caactgaaat cagagctggg acacagcgca ccagaagcta aagtcttgat gccatcaaag 2100gacatccctg ccccattcac atctctgtca cgtccactaa tcggcaaaag gagaaaagtg 2160agagaagatg acctaagtgt gactgcagca ggcagctctg gaaaatgaag ccagagcagt 2220gagccagccc ctcctccgac caaggaggaa ggaaagagca ggtaagcagg aaggccagtg 2280tcccagacag gaccctaatg atcctgaatc catgtatcag gatccatcct ctccttacac 2340ccttcctgga cacactctca cctctcattt tccaaagccc tgccatgctg ccatcccact 2400tccccacact ctgccccggg ttcccttttc ctaaagctgc agcttatggc ttctccagta 2460ggtggcagca cacacagagc cactcataaa ctgcagcttc tcagagcctg agagccagaa 2520accgtcccca gaaagtccct ccatagaaat aaaaaaacac cagaagtcac atttcatcct 2580tttacatggt tcccatctac cctcacaaca catgtcatca ccaaagacac acatacaagc 2640tccaatggct tttgccaggc aattcttcct ccaggacccc atctggcccc tccctcatcc 2700ctccccttgg actttgccct tcttactggc caggcagggg ggccagagtc caggcttgac 2760tcattcccac cttgtcctgg gctgagatcc caggtttgta acagaaaaca ccactaaagc 2820cccagcacag gagagaacca cccagcccag aagttccagg gaaggaactc tccggtccac 2880catggagtac ctctcagctc tgaaccccag tgacttactc aggtgactgc taaccctccg 2940ctctaccctc cacctttagg ggatataggc agggcaacaa tactccactc agcccctagg 3000agactaacca gtaccccttc ctctcctgct ccccactcca cagtgggctt gtcaagctcc 3060tgagccaccc gcccccacct gcactccatg gtctctccct catccctaat cgataaacct 3120agatctctcc ctccctagcc ctctagccac tctaccctca tcatgccctt tacactcacc 3180aagcccctcc tcgccccttc ttgacttttc ttctcaacta ccaggtcagt atctaatata 3240agctcggagt ttggacggag ggtctggacc tcagctccac caccccagcg acctttccgt 3300gtctgtgatc acaagcggac catccggaaa ggcctgacag ctgccacccg ccaggagctg 3360ctagccaaag taagtaggcc aagttcctcg gttcctatag caggggtagc caaggggctc 3420cacaacagtg gcaacttgtg atgatggagc agagggctga agtcacacag ctgcccctcc 3480ctctgagggc taaaagcagc ggagtgggcc taatgagctc tggtcaattt gttcattttc 3540cacctagtga gcttttctat gggagcaggg gttagcagga gatagggaga gttcgaggga 3600cggaattcag aagctagtat ggaaaggtga tttgtgtgac aaatcaagtt caaattctga 3660ttctgccact tcctgcctgt caaaccttgg gaagttgttc aacctaccaa aacctcagtt 3720tcttcaacta taaaaaggca ataataatac atcacctcct agggttgttg aaaggagtaa 3780gaggataatg taggtaaagt cctcatacct ggcacagagt aaggactcaa aaaggttaaa 3840cactattact gaaaacactt ctggagaact cttgagggtg tgggaagtga ggtgcagcat 3900tgtagataag acagaagggt ggacttcatg agaacctggc ttgctttcca attccaaacc 3960agaagtgact tggaggggag caaggggaga tgccaatgac atggtaggag caaagaggaa 4020aaaggtcagc ctctagctag gatcccccaa aaactgaaga acacggagag ctgcaacctt 4080taggaggtat caaagtgcca gaaagtcaaa gtgggacatc gaccaatgtc tagagccaac 4140tgatggatgt tgggcagcta aagagggaag gggcatggga taagacctgc ccttcttgct 4200tcttgccatt gggcaggcat tggagaccct actgctgaat ggagtgctaa ccctggtgct 4260agaggaggat ggaactgcag tggacagtga ggacttcttc cagctgctgg aggatgacac 4320gtgcctgatg gtgttgcagt ctggtcagag ctggagccct acaagggtaa gaggcctata 4380ctggggctgc ttccaatgcc tgtcctttag agctttcccg ggcttcctct ctagcttaac 4440cctgatcctg gggaccaggt gcaggaggag ttgtggaatt gtcaaggatg tcacacagtg 4500gacagaaagt ccaagcgagg gagggtctga cccagtgctg atggagatta gtggtgggtg 4560tctggtatga ggatctactg cactgacaag ggtgtcctac agagtggagt gctgtcatat 4620ggcctgggac gggagaggcc caagcacagc aaggacatcg cccgattcac ctttgacgtg 4680tacaagcaaa accctcgaga cctctttggc agcctgaatg tcaaagccac attctacggg 4740ctctactcta tgagttgtga ctttcaagga cttggcccaa agaaagtact caggtcagaa 4800atcaacatgt catactgccc catcccctac agttggatag tccccataat tcgtcctctt 4860gcacccacct acccctagtt agctcttgct tgtggaaagt cctcatctcc cagcttgatg 4920gcttcctccc aagttttcca aatcatctga tttcctcttg tctctgccat tcagggagct 4980ccttcgttgg acctccacac tgctgcaagg cctgggccat atgttgctgg gaatttcctc 5040cacccttcgt catgcagtgg agggggctga gcagtggcag cagaagggcc gcctccattc 5100ctactaaggg gctctgagct tctgccccca gaatcattcc aaccgaccca ctgcaaagac 5160tatgacagca tcaaatttca ggacctgcag acagtacagg ctagataacc cacccaattt 5220ccccactgtc ctctgatccc ctcgtgacag aacctttcag cataacgcct cacatcccaa 5280gtctataccc ttacctgaag aatgctgttc tttcctagcc acctttctag cctcccactt 5340gccctgaaag gccaagatca agatgtcccc caggcatctt gatcccagcc tgactgctgc 5400tacatctaat cccctaccaa tgcctcctgt ccctaaactc cccagcatac tgatgacagc 5460cctctctgac tttaccttga gatctgtctt catacccttc ccctcaaact aacaaaaaca 5520tttccaataa aaatatcaaa tatttaccac taagacttct gactccaatt taaaccagga 5580aagggatggg gtggataccc cattttgccc tcccccatca acacccagtc ccagatccaa 5640agcctcagtc ttcaagtatg gagttcaatg cccgcctccg cttggccacc gcaccctgct 5700gctgttccca agcctctcgc cgctttagga aggtagtcaa ggccacattt cgagccacat 5760ggtggccgaa agggtctctt atcagctcct ggttctgctc ccctgcaaag ggaaccactc 5820atgtttagta ctaccatgct caaagacaga ctctacttga cgtaaagatc cctccatttc 5880ccccagaccc tgggactgcc actgggataa agtgaacact tcactttcca aagaacagaa 5940gtcagagggc agggtgaggg cagaggacta cacaatcagt cagcgggagg gaaggggaaa 6000gcagactgct aagaacccat gagcaagacc accctttgga actaaagact gttgaatttc 6060agtagacttc ccagctaccc agtgatctgg agtaagagag gggagagtct ggaaacatca 6120aagagagggg ctggtactca ccaagctcag cagcaatttc cttccgggcc ctcaaggctg 6180ctccactcca gatggcatct agcacacggc tgccatggcg actacaggcc agagccacat 6240attgtccctg tatggagaca agcaaagtag gtctccctag caaaatccac aaaagtattt 6300tggaggcttg tggagagtga attttctgaa gagtttaagg gctgaattaa aatagtctag 6360gaattacata gcctttttga gtctgaaggt acagtccagg gaaagctcat attctctaaa 6420tggatgatgg tggggaatca aggcaaaagc cagaaatcta acctttaggt tctgcagcac 6480acggcggcgc acttgcgcgt cacagagggg ctggtcagga tggcatcgag cacatgagag 6540ccagcgggac tttgggcaag ggacagaagc tgtggtcccg tcaaggcacc cagacttcga 6600agtacaagac caggagtgga gaagtgcagc agatgctgga gcagtagaga cccaaggact 6660gtcacatccc ccaaggctct ggctgcggcc attgccacct ggcagcgcag agataagaaa 6720ttaggaggca ccaacaagcc aggtggatga gcccatcccc agaacactgc cacctccacc 6780tggatgaagt caccactctc acctcagaag tcagacgctt gctaaaccct gggaggcctg 6840actcccaatt agtcactggc ttggcctctc ccctacctca cctggtgctc tgcaggcact 6900gccccctcct cctccgtcag tccatagtac acctcataag ccatcaaagt ggcaaagaga 6960ggcacacagg ccacttgccg ggatgagggc tctgcacagt ggaatgccta aaggagaaga 7020caaaatatga tgaacctgag aacttctccg tcccttacct caactttaag ggaacacctt 7080caggaaagga aagaaggccc aactccacaa gaatgatagc tcyatgaaag cagatgtcat 7140gccttgctgt atccccagcc tctatcacat acaggggtta ataaatgttt atcccttaaa 7200taaacttctc tcttagttct gggagacaat ttttgttcca ggactgtgaa ataacatttg 7260gcctcagccg ggcacggtga gctgagattg cgccactgca ctccagcctg ggcaacaaga 7320gcaaaactcc gtctcaaaaa aaaaaaaaaa attagctggg tatggtggcg ggcacctata 7380ggcctcattt tcccccattc ctgtgaactc cttgggcatt agacttcctg agtccagagg 7440tacaaaaact taagattaga taagtcctaa aaggtagaag ttgaactgag ggcataaaca 7500gattgtgggt aatatccact cacctccaac aagagctgta ggaccttggc ttggtaggcc 7560ccaactctgc gacaggcccc caccagggca atgactaccc ctgggtggcc ctgggccaat 7620acagcttcca agacagggct cagctcctca aacacagggg acagctgatg ggaaacatgg 7680aatgtaactc tggcattctg gtaatggcgg accacattat tcagagaaat cttcccaacg 7740aaaacaacta aaaatgctag ataaaacata aaaagcattg ccagtatcag gacttttgtt 7800tggctagata aggcccagga taagggggca gtcttagagg tgccacctca caggaaggaa 7860ctttaagtgg ctcttcccta tgtgtaaggg agcactagga gcaaggggag actaaaatta 7920aactgcactt ccactctaac acccaatgca tgagaccaca gggaagccca ccttagtcct 7980aagcagcaaa ggaaaagaga agctatggca acagctctag cactcatgtg ggcttatagc 8040caagtaccag agtctgggtg ggccatggaa cctcaagctg taaacctgaa taaagccagt 8100ctcttgctgg tcctcatacc tgagagaagt caaaacaaat cctcactaga aggtgggcac 8160ctttatccta agctctggaa aacctcaaag aatagctttt caagggcaat ggccagcaca 8220atgtcaaagg tatatagagg cagacagacc ataaaataag gtccagtgat caagaattag 8280cagaaacaga tccagctcag gtttccaact caccagctca ggggtagtga ctgcatccag 8340taagcgctgc aaagggaagt tggcaatggg atgtgcagcc agggtctgca gctgcccctg 8400caagtgctcc tcaaagaggc tctggagtct tgggggctcc aacaccagca ggacctgctc 8460caggagtctg gaactcgtct gatctcggag aaatagcagt aggggactag ggacaagaag 8520ggcatcaggc agtcttaact ccatttcatg caaaatacag ttgcactaca tcacctcatt 8580catttcacac agccccgcca acttgggtcg atcctgaggc cctgaccata cctgccatct 8640actgaggaac cgcgagtact caggtagcca atcacagcat tgcagaggtg agcgcaaaac 8700tggggaagtt tgcggtgtaa aacctgtaaa gccacttgaa gacagaagct ggagatctta 8760tcagtgataa acactgtgga gagagggcag aaataaagcg gctttctcaa aatccagaca 8820acctgcgtaa cctcaaactt taggcaacaa gatacagggc actatggaca caaagggact 8880atacaaagtg ctctgaacgt caaaccagac caggtttgaa aagaagagga ataacctgag 8940gtaagggcct tgcttggtgg taagatgacc tccagacaag tagatacgag accatcctcc 9000tacttccctc cttacctgca atgtccttca gaaaggagga gctcaggtcc tgaaggcgat 9060tcaaaaaggt ttcagggact tcaaaatcag ctggcttaca ttcctgagct ggggtcttct 9120gtgcttctga aggaagatta agaacatgtg cagtggggag atcacaaact gccatccagg 9180agagaaaaat ttcacagatg aacaaggatt ccgcttggag atgcagaaca gatgtagtac 9240aactaaatgt ctcttctgga tgactgaact gagactacat ttctgtctta atttttctaa 9300gaaacttcaa ccagcttccc catggtccac agaagacagt tcaacctcct cagccaggta 9360ttcaagccat tccaaaataa agcctcattt aaactcgcta actttatttc ctagtttttc 9420aaaatgtgga tcacagctca agactgtacc atgaaatcaa gttagtggtc tcaactagca 9480ttttttttaa atgaaataga gcagaatagt tttgaaaagg aaaaaaaaat aacagagtgg 9540gctgcatgta gtaagggtat agtttcctga actyttgctt gagtccagtg tgtatgttac 9600ttttgcttaa gtccatttgt atattaagtc atgcttaaaa aaaattctcc tcctgcgtcc 9660attctaatag ccttccccaa acacaactgt acatccctac ctctgggcct ccgcacacac 9720taatctttgg cttgctaagc cctgttcaga ttaggacagg tcaagctaat tcttcccggt 9780caagctcccc catgaagact tactcttctg tttactgtct cattctgaac tttctaaatt 9840cttcttcccc ctaggtccac tttcctcttg taatacttac cagatgattg ggaaccacgg 9900ggcctggctc tctcagactc cagaatagtc cctcctaaca cctgaagcag agttctgacc 9960acgaagctgc catgtgtgtc tccacagtag acaagaaaat catcacacac ctcagcggct 10020agtcccagga ccagctcctc cagggtctcc gtgggaccat cctttccatc ctcctcctcc 10080tcctcctcct cctcctctgc agcactcccc agcaatcgag ggagctgtag caaagcactt 10140tgtaatacat ggaccccgca tcggtgacag gccacagtgc gcaagttaga gcgcagagca 10200gcccacacgc gacaaagcgg tttcaaggga ctgaatccca acagttcctg cagcatctca 10260ctgccagtcc tgttcgtgga caaagctagg gcctgagtct ctacttcctt cattatattg 10320tgcaccatca gatctgaaaa gggaagaaag acacactttg aagagactta gtagagaata 10380cagttatgta ccctcgctta accaagggtc gtgggtcgga ggccgaggaa gaggacatgg 10440gaaatgaaac taagtcccca cgccctgccg accctagaaa gtttccttgc ctgccctgga 10500tttgatgctc ttggtctaaa ccccgaagtt gctgcctacc tcgttcttcc ccagtctcgg 10560gagcctcttt caatgctgac agcgcccggc ggaaatatcc cagagcttcc gggctcaggt 10620gcgggtgcga atctggagcc ggctccgagc gcccatccgg aggcggccag ggttgccgct 10680tacggcctgg taaggggcgc cccgacccct tggccccgcg cccccgtttg ccaccagctg 10740ggaaccggcg ccccaccttg tgtggagagc gcggaccctg ccccatgtgt gcttcgcgac 10800ctgtccggct gcaaaagctt ccttaactgc ggacggacct tcgacgtcgc caagcgtgcg 10860ccttatccag acaaagttta gaaacgcggg cgcgctcctg acgcagctac tacgtcatag 10920ttccgcgccg ccccagccgg gcggggtggg tgtgtcaccc a 10961 2 1187 DNA Homosapiens 5′UTR 1..79 CDS 80..739 3′UTR 740..1187 polyA_signal 1158..1163AATAAA 2 agaaaacacc actaaagccc cagcacagga gagaaccacc cagcccagaagttccaggga 60 aggaactctc cggtccacc atg gag tac ctc tca gct ctg aac cccagt gac 112 Met Glu Tyr Leu Ser Ala Leu Asn Pro Ser Asp 1 5 10 tta ctcagg tca gta tct aat ata agc tcg gag ttt gga cgg agg gtc 160 Leu Leu ArgSer Val Ser Asn Ile Ser Ser Glu Phe Gly Arg Arg Val 15 20 25 tgg acc tcagct cca cca ccc cag cga cct ttc cgt gtc tgt gat cac 208 Trp Thr Ser AlaPro Pro Pro Gln Arg Pro Phe Arg Val Cys Asp His 30 35 40 aag cgg acc atccgg aaa ggc ctg aca gct gcc acc cgc cag gag ctg 256 Lys Arg Thr Ile ArgLys Gly Leu Thr Ala Ala Thr Arg Gln Glu Leu 45 50 55 cta gcc aaa gca ttggag acc cta ctg ctg aat gga gtg cta acc ctg 304 Leu Ala Lys Ala Leu GluThr Leu Leu Leu Asn Gly Val Leu Thr Leu 60 65 70 75 gtg cta gag gag gatgga act gca gtg gac agt gag gac ttc ttc cag 352 Val Leu Glu Glu Asp GlyThr Ala Val Asp Ser Glu Asp Phe Phe Gln 80 85 90 ctg ctg gag gat gac acgtgc ctg atg gtg ttg cag tct ggt cag agc 400 Leu Leu Glu Asp Asp Thr CysLeu Met Val Leu Gln Ser Gly Gln Ser 95 100 105 tgg agc cct aca agg agtgga gtg ctg tca tat ggc ctg gga cgg gag 448 Trp Ser Pro Thr Arg Ser GlyVal Leu Ser Tyr Gly Leu Gly Arg Glu 110 115 120 agg ccc aag cac agc aaggac atc gcc cga ttc acc ttt gac gtg tac 496 Arg Pro Lys His Ser Lys AspIle Ala Arg Phe Thr Phe Asp Val Tyr 125 130 135 aag caa aac cct cga gacctc ttt ggc agc ctg aat gtc aaa gcc aca 544 Lys Gln Asn Pro Arg Asp LeuPhe Gly Ser Leu Asn Val Lys Ala Thr 140 145 150 155 ttc tac ggg ctc tactct atg agt tgt gac ttt caa gga ctt ggc cca 592 Phe Tyr Gly Leu Tyr SerMet Ser Cys Asp Phe Gln Gly Leu Gly Pro 160 165 170 aag aaa gta ctc agggag ctc ctt cgt tgg acc tcc aca ctg ctg caa 640 Lys Lys Val Leu Arg GluLeu Leu Arg Trp Thr Ser Thr Leu Leu Gln 175 180 185 ggc ctg ggc cat atgttg ctg gga att tcc tcc acc ctt cgt cat gca 688 Gly Leu Gly His Met LeuLeu Gly Ile Ser Ser Thr Leu Arg His Ala 190 195 200 gtg gag ggg gct gagcag tgg cag cag aag ggc cgc ctc cat tcc tac 736 Val Glu Gly Ala Glu GlnTrp Gln Gln Lys Gly Arg Leu His Ser Tyr 205 210 215 taa ggggctctgagcttctgccc ccagaatcat tccaaccgac ccactgcaaa 789 * 220 gactatgacagcatcaaatt tcaggacctg cagacagtac aggctagata acccacccaa 849 tttccccactgtcctctgat cccctcgtga cagaaccttt cagcataacg cctcacatcc 909 caagtctatacccttacctg aagaatgctg ttctttccta gccacctttc tagcctccca 969 cttgccctgaaaggccaaga tcaagatgtc ccccaggcat cttgatccca gcctgactgc 1029 tgctacatctaatcccctac caatgcctcc tgtccctaaa ctccccagca tactgatgac 1089 agccctctctgactttacct tgagatctgt cttcataccc ttcccctcaa actaacaaaa 1149 acatttccaataaaaatatc aaatatttac cactaaga 1187 3 219 PRT Homo sapiens 3 Met Glu TyrLeu Ser Ala Leu Asn Pro Ser Asp Leu Leu Arg Ser Val 1 5 10 15 Ser AsnIle Ser Ser Glu Phe Gly Arg Arg Val Trp Thr Ser Ala Pro 20 25 30 Pro ProGln Arg Pro Phe Arg Val Cys Asp His Lys Arg Thr Ile Arg 35 40 45 Lys GlyLeu Thr Ala Ala Thr Arg Gln Glu Leu Leu Ala Lys Ala Leu 50 55 60 Glu ThrLeu Leu Leu Asn Gly Val Leu Thr Leu Val Leu Glu Glu Asp 65 70 75 80 GlyThr Ala Val Asp Ser Glu Asp Phe Phe Gln Leu Leu Glu Asp Asp 85 90 95 ThrCys Leu Met Val Leu Gln Ser Gly Gln Ser Trp Ser Pro Thr Arg 100 105 110Ser Gly Val Leu Ser Tyr Gly Leu Gly Arg Glu Arg Pro Lys His Ser 115 120125 Lys Asp Ile Ala Arg Phe Thr Phe Asp Val Tyr Lys Gln Asn Pro Arg 130135 140 Asp Leu Phe Gly Ser Leu Asn Val Lys Ala Thr Phe Tyr Gly Leu Tyr145 150 155 160 Ser Met Ser Cys Asp Phe Gln Gly Leu Gly Pro Lys Lys ValLeu Arg 165 170 175 Glu Leu Leu Arg Trp Thr Ser Thr Leu Leu Gln Gly LeuGly His Met 180 185 190 Leu Leu Gly Ile Ser Ser Thr Leu Arg His Ala ValGlu Gly Ala Glu 195 200 205 Gln Trp Gln Gln Lys Gly Arg Leu His Ser Tyr210 215 4 18 DNA Artificial Sequence sequencing oligonucleotide PrimerPU4 tgtaaaacga cggccagt 18 5 18 DNA Artificial Sequence sequencingoligonucleotide PrimerRP 5 caggaaacag ctatgacc 18

What is claimed:
 1. An isolated, purified, or recombinant polynucleotide comprising a contiguous span of at least 35 nucleotides of SEQ ID No 1 or the complements thereof.
 2. An isolated, purified, or recombinant polynucleotide comprising a contiguous span of at least 12 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least 1 of the following nucleotide positions of SEQ ID No 2: 1-78, 91-190, 208-229, 243-288, 301-328, 364-394, 409-457, 478-490, 505-508, 529-597, 616-633, 656-667, 682-688, 703-1188.
 3. An isolated, purified, or recombinant polynucleotide consisting essentially of a contiguous span of 8 to 50 nucleotides of SEQ ID No 1 or the complement thereof, wherein said span includes a CIDE B-related biallelic marker in said sequence.
 4. A polynucleotide according to claim 3, wherein said CIDE B-related biallelic marker is selected from the group consisting of the biallelic markers 12-73-49 and 12-74-38, and the complements thereof.
 5. A polynucleotide according to claim 3, wherein said contiguous span is 18 to 47 nucleotides in length and said biallelic marker is within 4 nucleotides of the center of said polynucleotide.
 6. A polynucleotide according to claim 5, wherein said polynucleotide consists of said contiguous span and said contiguous span is 25 nucleotides in length and said biallelic marker is at the center of said polynucleotide.
 7. A polynucleotide according to claim 5, wherein said polynucleotide consists essentially of a sequence selected from the following sequences: P(12-73-49) and P(12-74-38), and the complementary sequences thereto.
 8. A polynucleotide according to any one of claims 1, 2 or 3, wherein the 3′ end of said contiguous span is present at the 3′ end of said polynucleotide.
 9. A polynucleotide according to claim 3, wherein the 3′ end of said contiguous span is located at the 3′ end of said polynucleotide and said biallelic marker is present at the 3′ end of said polynucleotide.
 10. An isolated, purified, or recombinant polynucleotide consisting essentially of a contiguous span of 8 to 50 nucleotides of SEQ ID No 1 or the complement thereof, wherein the 3′ end of said contiguous span is located at the 3′ end of said polynucleotide, and wherein the 3′ end of said polynucleotide is located within 20 nucleotides upstream of a CIDE B-related biallelic marker in said sequence.
 11. A polynucleotide according to claim 10, wherein the 3′ end of said polynucleotide is located 1 nucleotide upstream of said CIDE B-related biallelic marker in said sequence.
 12. A polynucleotide according to claim 11, wherein said polynucleotide consists essentially of a sequence selected from the following sequences: D(12-73-49), D(12-74-38), E(12-73-49), and E(12-74-38).
 13. An isolated, purified, or recombinant polynucleotide consisting essentially of a sequence selected from the following sequences: B(12-73), B(12-74), C(12-73), and C(12-74).
 14. An isolated, purified, or recombinant polynucleotide which encodes a polypeptide comprising a contiguous span of at least 8 amino acids of SEQ ID No 3, wherein said contiguous span includes at least 1 of the following amino acid positions: 1-29, 47-70, 103-115, 124, 134, 169-185, and 203-219.
 15. A polynucleotide for use in a genotyping assay for determining the identity of the nucleotide at a CIDE B-related biallelic marker or the complement thereof.
 16. A polynucleotide according to claim 15, wherein the polynucleotide is used in an assay selected from the group consisting of a hybridization assay, a sequencing assay, an enzyme-based mismatch detection assay, and an amplification of a segment of nucleotides comprising said biallelic marker.
 17. A polynucleotide according to any one of claims 1-16 attached to a solid support.
 18. An array of polynucleotides comprising at least one polynucleotide according to claim
 17. 19. An array according to claim 18, wherein said array is addressable.
 20. A polynucleotide according to any one of claims 1-16 further comprising a label.
 21. A recombinant vector comprising a polynucleotide according to any one of claims 1-16.
 22. A host cell comprising a recombinant vector according to claim
 21. 23. A non-human host animal or mammal comprising a recombinant vector according to claim
 21. 24. A method of genotyping comprising determining the identity of a nucleotide at a CIDE B-related biallelic marker or the complement thereof in a biological sample.
 25. A method according to claim 24, wherein said biological sample is derived from a single subject.
 26. A method according to claim 25, wherein the identity of the nucleotides at said biallelic marker is determined for both copies of said biallelic marker present in said individual's genome.
 27. A method according to claim 24, wherein said biological sample is derived from multiple subjects.
 28. A method according to claim 24, further comprising amplifying a portion of said sequence comprising the biallelic marker prior to said determining step.
 29. A method according to claim 28, wherein said amplifying is performed by PCR.
 30. A method according to claim 24, wherein said determining is performed by a hybridization assay.
 31. A method according to claim 24, wherein said determining is performed by a sequencing assay.
 32. A method according to claim 24, wherein said determining is performed by a microsequencing assay.
 33. A method according to claim 24, wherein said determining is performed by an enzyme-based mismatch detection assay.
 34. A method according to any one of claims 24-33 wherein said CIDE B-related biallelic marker is selected from the group consisting of the biallelic markers 12-73-49 and 12-74-38 and the complements thereof.
 35. An isolated, purified, or recombinant polypeptide comprising a contiguous span of at least 8 amino acids of SEQ ID No 3, wherein said contiguous span includes at least 1 of the following amino acid positions: 1-29, 47-70, 103-115, 124, 134, 169-185, and 203-219.
 36. An isolated or purified antibody composition are capable of selectively binding to an epitope-containing fragment of a polypeptide according to claim 35, wherein said epitope comprises at least 1 of the following amino acid positions of SEQ ID No 3: 1-29, 47-70, 103-115, 124, 134, 169-185, and 203-219.
 37. A method for the screening of a candidate substance interacting with a CIDE B polypeptide comprises the following steps: a) providing a polypeptide comprising a contiguous span of at least 8 amino acids of SEQ ID No 3, wherein said contiguous span includes at least 1 of the following amino acid positions: 1-29, 47-70, 103-115, 124, 134, 169-185, and 203-219; b) obtaining a candidate substance; c) bringing into contact said polypeptide with said candidate substance; d) detecting the complexes formed between said polypeptide and said candidate substance.
 38. A method for screening molecules that modulate the expression of the CIDE B protein, said method comprising the steps of: a) cultivating a prokaryotic or an eukaryotic cell that has been transfected with a nucleotide sequence encoding said CIDE B protein or a variant or a fragment thereof, placed under the control of its own promoter; b) bringing into contact the cultivated cell with a molecule to be tested; c) quantifying the expression of said CIDE B protein or a variant or a fragment thereof.
 39. A method for screening of a candidate substance or molecule that modulated the expression of the CIDE B gene, said method comprising the steps of: a) providing a recombinant cell host containing a nucleic acid, wherein said nucleic acid comprises the 5′ regulatory region of CIDE B or a biologically active fragment or variant thereof located upstream a polynucleotide encoding a detectable protein; b) obtaining said candidate substance, and c) determining the ability of said candidate substance to modulate the expression levels of the polynucleotide encoding the detectable protein.
 40. A diagnostic kit comprising a polynucleotide according to any one of claims 1-16. 